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DRUG METABOLISM AND DISPOSITION
Copyright © 2001 by The American Society for Pharmacology and Experimental Therapeutics
DMD 29:232–241, 2001
Vol. 29, No. 3, Part 2
214/882731
Printed in U.S.A.
INVESTIGATION OF THE IN VITRO METABOLISM PROFILE OF A
PHOSPHODIESTERASE-IV INHIBITOR, CDP-840: LEADING TO STRUCTURAL
OPTIMIZATION
CHUN LI, NATHALIE CHAURET, LAIRD A. TRIMBLE, DEBORAH A. NICOLL-GRIFFITH, JOSE M. SILVA,
DWIGHT MACDONALD, HELENE PERRIER, JAMES A. YERGEY, TED PARTON, RAKKI P. ALEXANDER,
AND GRAHAM J. WARRELLOW
Department of Medicinal Chemistry, Merck Frosst Center for Therapeutic Research, Pointe-Claire-Dorval, Quebec, Canada (C.L., N.C., L.A.T.,
D.A.N.-G., J.M.S., D.M., H.P., J.A.Y.); and Celltech Chiroscience Ltd., Slough, Berkshire, United Kingdom (T.P., R.A., G.W.)
Received August 18, 2000; accepted November 8, 2000
This paper is available online at http://dmd.aspetjournals.org
CDP-840 is a selective and potent phosphodiesterase type IV inhibitor, whose in vitro metabolism profile was first investigated
using liver microsomes from different species. At least 10 phase I
oxidative metabolites (M1–M10) were detected in the microsomal
incubations and characterized by capillary high-performance liquid chromatography continuous-flow liquid secondary ion mass
spectrometry (CF-LSIMS). Significant differences in the microsomal metabolism of CDP-840 were found between rat and other
species. The major route of metabolism in rat involved para-hydroxylation on the R4 phenyl. This pathway was not observed in
human and several other species. The in vitro metabolism profile of
CDP-840 was further examined using freshly isolated hepatocytes
from rat, rabbit, and human. The hepatocyte incubations indicated
more extensive metabolism relative to that in microsomes. In ad-
dition to the phase I oxidative metabolites observed in microsomal
incubations, several phase II conjugates were identified and characterized by CF-LSIMS. Interspecies differences in phase II metabolism were also found in these hepatocyte incubations. The
major metabolite in human hepatocytes was identified as the pyridinium glucuronide, which was not detected in rat hepatocytes.
Simple structural modification on R4, such as p-Cl substitution,
greatly reduced the species differences in microsomal metabolism. Furthermore, modifications on R3, such as the N-oxide, eliminated the N-glucuronide formation in human. These results not
only helped in determining the suitability of animal species used in
the preclinical safety studies but also provided valuable directions
for the synthetic efforts in finding backup compounds that are
more metabolically stable.
Cyclic phosphodiesterase (PDE1) enzymes catalyze the hydrolysis
of the 3⬘-phosphoester bonds of cAMP and cGMP to form the corresponding AMP and GMP, and therefore, are involved in controlling
the intracellular concentrations of cAMP and cGMP (Torphy, 1998).
At least 11 mammalian PDE isozyme families have been reported,
each encoded by a distinct gene. They are distinguished on the basis
of their enzyme kinetics, substrate selectivity, and tissue distribution.
The type IV family of phosphodiesterases (PDE-IV) is a high-affinity
cAMP-selective isozyme, and has been found in almost all cell types
that have been implicated in asthma pathogenesis (O’Brien, 1997).
Selective PDE-IV inhibitors, therefore, could become promising therapeutic agents for the treatment of asthma and a wide range of other
inflammatory diseases (Torphy et al., 1994). CDP-840 {R-[⫹]-4-[2-
(3-cyclopentyloxy-4-methoxy phenyl)-2-phenyl ethyl] pyridine} is a
potent and selective PDE-IV inhibitor (Hughes et al., 1997; Perry et
al., 1998), and was in development for the treatment of asthma. The
structure of CDP-840 is shown in Fig. 1.
Drug metabolism studies during the early drug discovery stage are
becoming increasingly important. They provide information useful for
many aspects of drug discovery, such as drug design, pharmacokinetic
evaluation, and toxicity assessment. Biotransformations can be performed in vitro with the microsomal or cytosolic fraction of liver
tissue as the enzyme source under the appropriate incubation conditions. Microsomal incubations are useful to pinpoint specific pathways (oxidation or glucuronidation), and can be used to gain an
understanding of potential interspecies differences in metabolism
(Kumar et al., 1999). Hepatocyte incubations retain phase I and phase
II enzyme activities, and therefore, are useful to determine overall
metabolism, and mimic in vivo metabolism more accurately than
incubations with subcellular fractions (Placidi et al., 1997). In vitro
incubations with liver microsomes and/or hepatocytes can be used as
a means to predict potential biotransformations in humans and in
those species used for preclinical safety studies.
Identification of metabolic pathways of drug candidates significantly relies on innovations in analytical chemistry. The role of mass
spectrometry, especially liquid chromatography/mass spectrometry
(LC/MS), in drug metabolite identification has become evident and
1
Abbreviations used are: PDE, phosphodiesterase; CDP-840, R-[⫹]-4-[2-(3cyclopentyloxy-4-methoxy phenyl)-2-phenyl ethyl] pyridine; LC/MS, liquid chromatography/mass spectrometry; HPLC, high-performance liquid chromatography; CF-LSIMS, continuous-flow liquid secondary ion mass spectrometry;
UDPGA, uridine 5⬘-diphosphate glucuronic acid; MS/MS, mass spectrometry/
mass spectrometry; amu, atomic mass unit.
Send reprint requests to: Chun Li, Medicinal Chemistry Dept., Merck Frosst
Centre for Therapeutic Research, P.O. Box 1005, Pointe-Claire-Dorval, Quebec,
H9R 4P8, Canada. E-mail: [email protected]
232
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ABSTRACT:
IN VITRO METABOLISM STUDIES OF PDE-IV INHIBITORS
233
NMR analysis and comparison to authentic standards wherever possible. Based on the in vitro metabolism results obtained with CDP840, several backup compounds with simple structural modifications
were evaluated, such as p-Cl substitution on R4 phenyl (CT2412) and
N-oxide on R3 (CT2481) (Fig. 1). These simple modifications significantly improved the metabolism profile and metabolic stability. Our
in vitro metabolism studies proved to be extremely useful, not only for
predicting in vivo metabolism in animal models and in humans but
also for guiding the medicinal chemistry efforts for structural optimization of the lead compound CDP-840.
Experimental Procedures
The substructures are labeled as R1 (cyclopentyl), R2 (catechol phenyl ring), R3
(pyridyl), and R4 (phenyl).
increasingly important in recent years (Vrbanac et al., 1992; Iwabuchi
et al., 1994; Davis and Baillie, 1995; Jackson et al., 1995). Capillary
HPLC/continuous-flow liquid secondary ion mass spectrometry (CFLSIMS) is a powerful LC/MS technique for metabolite identification
(Moritz et al., 1992; Onisko et al., 1994), and has been used effectively and routinely in our laboratory for metabolism studies (NicollGriffith et al., 1993; Li et al., 1995). In this article, the metabolic
profiles of CDP-840 were investigated using liver microsomes and
hepatocytes prepared from different species. The metabolites were
rapidly characterized by CF-LSIMS, supplemented with the use of
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FIG. 1. Chemical structure of CDP-840, R-[⫹]-4-[2-(3-cyclopentyloxy-4-methoxy
phenyl)-2-phenyl ethyl]pyridine, CT2412, and CT2481.
Materials. CDP-840 (Warrellow et al., 1997, example 16), CT2412,
CT2481 were synthesized at Celltech (Slough, UK). All synthetic metabolite
standards were synthesized at Celltech or Merck Research laboratories. The
synthesis and characterization of some of the metabolites were described
(Warrellow and Alexander, 1996; Warrellow et al., 1997). NADP (Na⫹ salt)
and glucose-6-phosphate dehydrogenase were purchased from Sigma Chemical Co. (St. Louis, MO). All solvents used were obtained from commercial
sources and were of HPLC grade.
Microsomal Oxidative Incubations. Hepatic microsomes were prepared
from frozen livers (human, rat, rabbit, ferret, rhesus monkey, etc.) according to
a standard procedure (Lu and Levin, 1972). Incubations with microsomes
prepared from different species were typically conducted under linear conditions with 80 ␮M CDP-840 (or CT2412, CT2481) and 0.5 mg of microsomal
protein in the presence of an NADPH-generating system as previously described (Nicoll-Griffith et al., 1993). Incubations (0.5 ml) were conducted for
30 min at 37°C and were quenched by the addition of an equal volume of
acetonitrile. Precipitated proteins were removed by centrifugation (10,000
rpm; Eppendorf centrifuge model 5415C) for 10 min. Supernatants were
analyzed by HPLC/UV and HPLC/CF-LSIMS. Blank incubations containing
no drug and control incubations containing boiled microsomes were also
conducted at the same time.
Microsomal Glucuronidation. Glucuronidation was achieved using an
incubation of 200 ␮M CDP-840 (or CT2412, CT2481) with 1.0 mg of
microsomal protein for 30 min. The incubation was conducted with 12.5 mM
MgCl2, 12.5 mM uridine 5⬘-diphosphate glucuronic acid (UDPGA) and 20
mM D-saccharic acid-1,4-lactone in a phosphate buffer at pH 6.6. The incubation mixture (0.5 ml) was quenched with an equal volume of acetonitrile.
Precipitated proteins were removed by centrifugation for 10 min, and supernatants were analyzed by HPLC/UV and HPLC/CF-LSIMS.
Hepatocyte Incubations. Rat hepatocytes were isolated from male
Sprague-Dawley rats by collagenase perfusion of liver as described previously
(Silva et al., 1998). Fresh human liver tissue was obtained from consenting
donors undergoing partial hepatectomies and from unused liver portions from
patients undergoing liver transplants (St. Luc Hospital, Montreal, Canada). The
tissues used were morphologically healthy. Human hepatocyte isolation was
conducted by a two-step collagenase perfusion of the liver sample as described
by Silva et al. (1998). Rabbit hepatocytes were prepared in a similar way to the
human hepatocytes. Incubations were conducted with 50 ␮M CDP-840
(CT2412, CT2481) and 2 ⫻ 106 isolated hepatocyte cells/ml. The cell mixture
was incubated in Krebs-Henseleit buffer containing 12.5 mM HEPES (pH ⫽
7.4) at 37°C under 95% air and 5% CO2 for 3 h, and was quenched by the
addition of an equal volume of acetonitrile. Precipitated proteins were removed
by centrifugation for 10 min. Supernatants were analyzed by HPLC/UV and
HPLC/CF-LSIMS.
In Vivo Pharmacokinetics and Metabolism. CDP-840 and CT2412 were
administered p.o. at 20 mg/kg in 1% methocel (pH ⫽ 2.0) to rats, and were also
administered i.v. at 5 mg/kg in saline to rats. CDP-840 was dosed p.o. at 10
mg/kg to rabbits, and 15 mg b.i.d. to humans (Harbinson et al., 1997). The
plasma samples obtained postdosing were quenched with an equal volume of
acetonitrile, and analyzed by HPLC/UV. Selected samples were analyzed by
LC/MS.
Isolation of Metabolites. To prepare ⬃0.1 mg of metabolites M12 and M17
for NMR characterization, human microsomal incubations in the presence of
UDPGA were scaled up appropriately. The isolation was similar to that
described previously (Li et al., 1995), using a preparative Waters Novapak C18
234
LI ET AL.
TABLE 1
Extent of metabolism of CDP-840 and its analogs in microsomes and
hepatocytes
Rate of Metabolism (N ⫽ 2)a
Incubation
Rat microsomes
Rhesus monkey microsomes
Mouse microsomes
Rabbit microsomes
Ferret microsomes
Guinea pig microsomes
Dog microsomes
Human microsomes
(pooled)
Male rat hepatocytes
Rabbit hepatocytes
Human hepatocytes
CDP-840
CT2412
CT2481
0.88
0.75
0.27
0.40
0.27
0.48
0.13
0.53
0.48
1.04
0.45
1.23
7.7
4.5
4.2
a
Rate of metabolism is expressed as nanomoles per minute per milligram protein for
microsomal incubations, and nanomoles per hour per 106 cells.
Results
Oxidative Microsomal Metabolism of CDP-840. The in vitro
metabolism profiles of CDP-840 in humans and potential safety
animal species were investigated using microsomal preparations. The
incubation mixtures of CDP-840 with microsomal proteins from various species under oxidative conditions were first analyzed by analytical HPLC with UV-photodiode array detection. The percentage of
metabolism was calculated using the LC/UV peak area ratio of CDP840 in an incubation with a control incubation (without NADPH). The
rate of microsomal metabolism (nmol/min 䡠 mg) of CDP-840 in
different species is summarized in Table 1. The UV spectra of the
FIG. 2. Reconstructed ion chromatograms (sum of MH⫹ of CDP-840 and its
metabolites) that represent the metabolism profiles of CDP-840 in incubations
from human microsomes (a), rabbit microsomes (b), and rat microsomes (c).
metabolites were similar to that of CDP-840, except for one metabolite, M5. LC/UV profiles of these microsomal incubations presented
not quantitative but relative percentages of most of the metabolites
generated. The incubation samples were also analyzed using capillary
HPLC CF-LSIMS, which did not have UV on-line. The total ion trace
has high background consisting of glycerol adduct ions (Li et al.,
1995), because glycerol was added to the mobile phase and used as
matrix for CF-LSIMS. Reconstructed ion chromatograms of the molecular ions for the parent and all metabolites were therefore presented, as shown in Fig. 2. The reconstructed positive ion chromatograms showed very similar percentage of each metabolite compared
with the analytical HPLC/UV traces. The microsomal metabolism
profile of CDP-840 in rat differed significantly from other species. At
least 10 different metabolites were detected, named M1 through M10.
The LSI mass spectra of CDP-840 and three representative phase I
metabolites, M5, M6, and M9, are shown in Fig. 3, a– d, and the
fragmentation patterns of phase I metabolites are summarized in Table
2.
The positive ion primary mass spectrum of CDP-840 (Fig. 3a)
displayed an intense molecular ion [M ⫹ H]⫹ at m/z 374 and a minor
glycerol adduct [M ⫹ H]⫹ at m/z 466. Three unique fragment ions, A,
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column (7.5 ⫻ 300 mm), and a Waters 990 diode array detector. Separation of
metabolites and parent compound was carried out using a linear gradient of
60% A to 90% A (A ⫽ CH3OH; B ⫽ 20 mM NH4OAc, pH 5.0) in 30 min at
a flow of 4 ml/min. Metabolite peaks were collected manually, concentrated,
and desalted using 1 ml of BondElut C18 SPE cartridges, and dried in a
Heto-Vac CT110 vacuum centrifuge (Heto Lab Equipment, Berkerod, Denmark).
CF-LSIMS. A JEOL HX110A double focusing mass spectrometer (EB
configuration; JEOL, Boston. MA), equipped with a 10-kV LSIMS source and
a cesium ion gun was used in this study. The mass spectrometer was operated
in the CF-LSIMS mode, and was described in detail previously (Li et al.,
1995). Data acquisition was in positive ion mode and the mass spectrometer
was scanned at a rate of 4 s from m/z 0 to 1000 Da. Separation of CDP-840 and
its metabolites was achieved using a KAPPA Hypersil BDS C18 (0.30 ⫻
100-mm) capillary column (Keystone Scientific, Bellefonte, PA). The flow
rate was 3 ␮l/min, which was obtained by splitting the main flow (1 ml/min)
using a Valco tee (Valco Instruments, Houston, TX). A linear gradient was
used from 50% A to 90% A over 40 min (A ⫽ methanol, B ⫽ 20 mM
ammonium acetate adjusted to pH 5.0 with acetic acid; each solvent contained
1.5% glycerol). The incubation supernatant was diluted 5-fold with aqueous
mobile phase, and 10 ␮l was injected onto the capillary HPLC column. The
capillary column was flushed with 100% methanol whenever not used for
analysis.
MS/MS analysis was carried out using a B/E linked scan. The collision gas
used was helium and the pressure was adjusted such that the intensity of the
precursor ion was reduced by 50%.
NMR Characterization of Glucuronide Conjugates M12 and M17.
NMR spectra were acquired at 22°C on a Bruker AMX 500 equipped with a
5-mm inverse broad-banded probehead. Samples were dissolved in 160 ␮l of
dimethyl sulfoxide-d6 and placed in a Shigemi symmetrical microtube matched
to the solvent. One-dimensional 1H and two-dimensional double quantumfiltered correlation spectra, heteronuclear multiple quantum correlation spectra, and heteronuclear multiple bond correlation spectra were acquired with
standard parameters.
235
IN VITRO METABOLISM STUDIES OF PDE-IV INHIBITORS
TABLE 2
Compound
FIG. 3. CF-LSI background-subtracted mass spectra of CDP-840 (a), M5 (b),
M6 (c), and M9 (d).
CDP-840
M1
M2
M3, M8
374
360
390
390
M4, M7, M9
M5
M6
M10
390
390
390
306
M6⬘
M11
M12
M14, M15
M17
M18
M19
M20
420
536
550
566
566
596
386
697
a
B, and C, at m/z 281, 213, and 182 were detected. These three
fragment ions represented the sequential elimination of methyl pyridyl, cyclopentyl, and methoxy moieties from the protonated molecule
(Table 2). Collision-induced dissociation of the molecular ion (B/E
linked scan) also produced the same fragment ions (data not shown),
and therefore, did not offer any additional structural information.
These characteristic fragmentations were subsequently used to determine which of the substructures had undergone metabolism.
The mass spectrum of M1 showed an abundant molecular ion at m/z
360, which is 14 amu less than that of CDP-840. Two diagnostic
fragment ions (A and B) at m/z 267 and 199 were also shifted 14 amu
lower relative to the fragment ions of CDP-840. This implied that M1
was an O-desmethyl metabolite.
The primary LSI mass spectra of M2 to M9 all showed abundant
molecular ions at m/z 390, i.e., 16 amu higher than that of CDP-840,
indicating that they are different mono-oxygenated metabolites. The
mass spectrum of M2 showed fragment ions A and B at m/z 297 and
229, also 16 amu higher than the corresponding fragments 281 and
213 of CDP-840, suggesting that the methyl pyridyl and cyclopentyl
moieties were not altered. The exact site of hydroxylation could not be
assigned based on its LSI mass spectrum or MS/MS spectrum. M2
was speculated to be the monohydroxy metabolite on the C2 position
of the ethyl link (Fig. 9), based on its coelution and identical mass
spectrum with an available authentic standard (racemic). The chirality
of this metabolite was not determined.
m/z of Fragment Ions
MH⫹
Present in
373
373
374
360
374
390
390
420
306
390
A
B
C
281
267
297
281
213
199
229
213
182
182
198
182
297
281
297
213
213
213
229
182
182
198
182
327
267
281
297
297
327
213
297
259
199
213
213
229
259
229
182
182
182
198
228
198
All species investigateda
All species investigateda
All species investigated
except rata
All species investigateda
All species investigateda
Rat, rabbit
All species investigated
except rat
Rat
Rat, rabbit
Rabbit, human
Rabbit
Rat, rabbit
Rat, rabbit
Rabbit
Rat
Rhesus monkey, rat, human, rabbit, guinea pig, dog, mouse, and ferret.
The primary LSI mass spectra of M3 and M8 were identical, both
showed fragment ions A and B at m/z 281 and 213, suggesting that
oxidation occurred on the methyl pyridyl moiety. M8 was confirmed
as a monohydroxy metabolite on the C1 position of the ethyl link (Fig.
9), based on coelution and similar mass spectrum with an authentic
standard (chiral with C1: S and C2: R). M3 was likely to be the
diastereoisomer of M8. A notable minor fragment ion at m/z 373 was
present in the primary mass spectra of M2, M3, and M8, but was not
present in the corresponding product ion MS/MS spectra (B/E linked
scan) of [M ⫹ H]⫹. This fragment ion is postulated to arise from the
loss of OH radical from [M ⫹ H]⫹ by the initial high-energy ionization process, and seemed to be diagnostic of these two types of
hydroxylation.
The primary mass spectrum of M4 showed strong fragment ions A
and B at m/z 297 and 213, indicating that the hydroxylation was on the
R1 cyclopentyl group. The exact position of hydroxylation and the
stereochemistry could not be determined based on its mass spectrum.
Similar mass spectra were obtained for M7 and M9 (Fig. 3d), suggesting that they were either positional isomers or diastereoisomers of
M4.
In the primary mass spectrum of M5 (Fig. 3b), fragment ions A and
B at m/z 281 and 213 suggested that oxidation was on the methyl
pyridyl moiety. Another abundant fragment ion at m/z 374 was also
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Mass spectral fragmentation pattern of CDP-840 and its metabolites
236
LI ET AL.
FIG. 4. Reconstructed ion chromatograms (sum of MH⫹ of CDP-840 and its
metabolites) that represent the metabolism profiles of CDP-840 in incubations
from rat hepatocytes (a), rabbit hepatocytes (b), and human hepatocytes (c).
tion. The primary LSI mass spectra exhibited abundant molecular ions
[M ⫹ H]⫹ at m/z 566. The mass spectrum of M15 (Fig. 5b) below
mass 400 amu resembled that of M9, the monohydroxy cyclopentyl
metabolite. M15 was postulated to be the glucuronide conjugate of a
hydroxy cyclopentyl, although the possibility of a hydroxy cyclopentyl, pyridinium glucuronide could not be ruled out. M14 was very
minor, and its mass spectrum was too weak to produce useful fragment ions. It was likely an isomer of M15.
Metabolite M19 was also formed only in the rabbit hepatocyte
incubation. Ions at m/z 386 and 403 corresponded to [M ⫹ H]⫹ and
[M ⫹ NH4]⫹. Fragment ions at m/z 213 and 306 were detected,
suggesting that the cyclopentyl group was lost. The mass difference
between [M ⫹ H]⫹ 386 and fragment ion 306 was 80, implying a
sulfate conjugate (Fig. 10).
Several metabolites such as M6⬘, M13, M16, M17, M18, and M20
were detected in the rat hepatocyte incubation, but not in the human
hepatocyte incubation. The mass spectrum of M17 showed abundant
molecular ions [M ⫹ H]⫹ at m/z 566. The spectrum below mass 400
amu was identical to that of M6, the para-phenol metabolite. M17
was, therefore, identified as R4 para-hydroxy glucuronide conjugate
(Fig. 5c).
The mass spectrum of M13 (data not shown) displayed a strong [M
⫹ H]⫹ at m/z 408, 34 amu higher than that of CDP-840. Fragment
ions at m/z 390, 297, and 229 were detected, consistent with a
dihydrodiol on the R4 phenyl. The mass spectrum of M16 (data not
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detected. This fragment is characteristic of an N-oxide, and was
produced by the high-energy ionization process, but was not present
in the MS/MS spectrum of [M ⫹ H]⫹. Metabolite M5 was confirmed
as the R3 N-oxide with the authentic standard. This metabolite was the
only one which showed different UV spectrum with that of CDP-840.
The extinction coefficient for the N-oxide is about 3 times greater than
that of CDP-840. When a standard solution containing equal concentrations of the N-oxide and CDP-840 was analyzed by CF-LSIMS, the
intensity of MH⫹ of the N-oxide (m/z 390) was about half of the MH⫹
of CDP-840 (m/z 374), but the N-oxide also produced a fragment ion
at m/z 374 with equal intensity with MH⫹ at m/z 390. When the
extracted ion chromatograms for 374 and 390 were summed, the
resulting reconstructed ion chromatogram showed nearly the same
response for the two compounds. Therefore, we believe that the
reconstructed CF-LSIMS profiles shown in Fig. 2 represented the
relative amount of metabolites generated.
The primary LSI mass spectrum of M6 (Fig. 3c), a major metabolite
in the rat microsomal incubation, showed abundant fragment ions at
m/z 229 and 197, similar to that of M2, except for the absence of a
fragment ion at m/z 373. This metabolite was confirmed as the R4
para-phenol, as it coeluted with and showed a mass spectrum identical
to the authentic standard.
The LSI mass spectrum of M10 showed an abundant molecular ion
[M ⫹ H]⫹ at m/z 306, 68 amu less than that of CDP-840. Fragment
ion A at m/z 213 represented the loss of methyl pyridyl from [M ⫹
H]⫹. The spectrum was consistent with the O-descyclopentyl metabolite, and was confirmed using an authentic standard.
Metabolism of CDP-840 in Hepatocytes. Incubations of CDP-840
were carried out with hepatocytes isolated from rat, rabbit, and human
livers. The reconstructed positive ion chromatograms (summed MH⫹
of CDP-840 and all metabolites) are shown in Fig. 4. The rate of
metabolism (nmol/h 䡠 106 cells) in these hepatocyte incubations was
summarized in Table 1. Both phase I oxidative metabolites (M1–M10)
and phase II conjugates were detected. The formation of the phase II
metabolites differed significantly between the three species. These
phase II metabolites were characterized by CF-LSIMS, and in some
cases were also confirmed by NMR. The mass spectral fragmentation
patterns of these phase II metabolites are summarized in Table 2.
M11 was present in the rat and rabbit hepatocyte incubations, but
not in the human hepatocyte incubation. The primary LSI mass
spectrum of M11 showed an abundant molecular ion [M ⫹ H]⫹ at m/z
536. Fragment ions at m/z 360, 267, and 199 were observed, and the
spectrum below m/z 400 was identical to that of M1, the O-desmethyl
metabolite. The mass difference between [M ⫹ H]⫹ (536) and 360 is
176 (dehydrated glucuronic acid), consistent with a glucuronide conjugate. The exact site of glucuronide attachment (either an O-linked or
N-linked glucuronide) could not be determined from the LC/MS or
LC/MS/MS spectrum.
M12 was detected in both rabbit and human hepatocytes; in fact it
was the major metabolite in human hepatocytes. However, rat hepatocytes did not produce M12. The primary LSI mass spectrum of M12
(Fig. 5a) showed an abundant molecular ion at m/z 550. The spectrum
below m/z 400 was very similar to that of CDP-840, with fragment
ions at m/z 374, 281, and 213. The mass difference between molecular
ion (550) and 374 is 176, consistent with a glucuronide conjugate.
High-energy collision-induced dissociation of [M ⫹ H]⫹ at m/z 550
also produced similar fragment ions (data not shown). The pyridine
nitrogen is the only functional group in the CDP-840 structure to
which a glucuronide could be directly linked; therefore, it was proposed that M12 was a pyridinium glucuronide based on its mass
spectrum.
M14 and M15 were present only in the rabbit hepatocyte incuba-
IN VITRO METABOLISM STUDIES OF PDE-IV INHIBITORS
237
FIG. 6. Structure of M12, pyridinium glucuronide, as determined by NMR.
shown) showed a molecular ion [M ⫹ H]⫹ at m/z 406, 32 amu higher
than that of CDP-840, indicating a dioxygenated metabolite, and was
proposed to be an R4 catechol.
Metabolite M6⬘, which eluted very closely with M6, showed a
protonated molecule at m/z 420, and major fragment ions at m/z 327
and 259. The LSI mass spectrum of M6⬘ exhibited a very similar
pattern to that of M6, except that all the ions were shifted 30 amu
higher. The increment of 30 amu could imply an additional methoxy
group on the R4. This metabolite was proposed to be the R4 O-methyl
catechol.
Metabolite M18 showed abundant molecular ion at m/z 596, the LSI
mass spectrum exhibited a very similar pattern to that of M17, except
that all the ions were shifted 30 amu higher. The increment of 30 amu
would again imply an additional methoxy group on the R4 phenyl (see
structure in Fig. 10).
The LSI mass spectrum of metabolite M20 displayed a strong [M ⫹
H]⫹ at m/z 697. Fragment ions at m/z 229, 281, 297, 374, and 390
were detected. The spectrum suggested that M20 was a glutathione
adduct (Fig. 5d).
Glucuronidation of CDP-840 in Microsomes. A proposed pyridinium glucuronide metabolite M12 was detected in rabbit and human
hepatocytes. If the proposed structure is correct, the same metabolite
might be formed in the incubation of CDP-840 with microsomal
proteins from various species in the presence of UDPGA. Indeed,
M12 was detected in the incubations with human, rabbit, rhesus
monkey, and guinea pig microsomes in the presence of UDPGA at
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 18, 2017
FIG. 5. CF-LSI background-subtracted mass spectra of phase II metabolites M12
(a), M15 (b), M17 (c), and M20 (d).
rates of 1.23, 0.17, 0.03, and 0.02 nmol/min 䡠 mg of protein. This
metabolite was not detected in the incubations with rat, dog, mouse,
or ferret microsomes, nor was it not detected in the rat hepatocyte
incubation.
M12 was isolated from the human microsomal incubation by semipreparative HPLC. The structure was identified as ␤-linked pyridinium glucuronide (Fig. 6; Table 3) by NMR. The linkage between
the glucuronide anomeric carbon and the pyridyl nitrogen was confirmed by long-range correlation experiments. In particular, a threebond correlation between the glucuronide anomeric proton (H-26) and
the two carbons adjacent to the pyridyl nitrogen (C-11 and C-12) was
observed. In addition, the reverse correlation between the two protons
adjacent to the pyridyl nitrogen (H-11 and H-12) and the glucuronide
anomeric carbon (C-26) was also observed, confirming the presence
of the pyridinium glucuronide species depicted in Fig. 6. A coupling
constant of 8.6 Hz was observed at the anomeric proton, indicating
that the glucuronide linkage has the ␤ configuration.
When the authentic standard of M6 (R4 para-phenol) was incubated with rat microsomal protein in the presence of UDPGA, a
metabolite was also detected. This metabolite had the same retention
time and LSI mass spectrum as those of M17 in rat hepatocytes. M17
was isolated from the microsomal incubation by prep-HPLC, and
NMR studies confirmed its structure as R4 para-O-glucuronide with
the configuration.
In Vitro Metabolism of CT2412 and CT2481. CT2412 and
CT2481 are two synthetic analogs (see structures in Fig. 1) of CDP840. Both have a para-Cl substitution on the R4 phenyl, and CT2481
also has a simple modification on R3, i.e., N-oxide. The in vitro
metabolism profiles of these two analogs were investigated using
microsomal preparations from rat and rhesus monkey. The microsomal oxidative metabolism profiles of CT2412 in rat and rhesus monkey are shown in Fig. 7. The profiles were clearly similar in the two
species. The metabolites were identified analogously to those described for CDP-840 metabolites. They were descyclopentyl (1),
hydroxy cyclopentyl (2 and 3), N-oxide (4), and desmethyl (5) metabolites. When CT2412 was incubated with microsomes under glucuronidation conditions, the N-glucuronide metabolite of CT2412 was
detected in human microsomes (3%) and rabbit microsomes (3%), but
to a lesser extent compared with that of CDP-840. The microsomal
oxidative metabolism profiles of CT2481 in rat and rhesus monkey
were also identical (data not shown), with descyclopentyl, hydroxy
cyclopentyl, and desmethyl being the major metabolites. The reduction of N-oxide to free pyridyl was also observed, but was very minor
based on LC/UV and LC/MS data. In the glucuronidation studies, the
238
LI ET AL.
TABLE 3
1
H chemical shifts of CDP-840 and its glucuronide metabolite M12
CDP-840
Position
␦a
Glucuronide Metabolite M12
Multiplet
J
ppm
a
6.87
6.79
6.79
4.46
3.69
3.64
7.86
8.71
7.35
7.25
7.14
4.73
1.85–1.50
3.64
Hz
s
s
s
t
dd
dd
d
d
d
t
t
m
m
s
8.5
14.0,8.5
14.0,8.5
6.6
6.6
7.8
7.8
7.8
Multiplet
ppm
6.93
6.81
6.81
4.51
3.77
3.72
8.01
8.94
7.36
7.27
7.15
4.75
1.87–1.49
3.66
5.51
3.37
3.31
3.47
3.30
J
Hz
s
s
s
t
dd
dd
d
d
d
t
t
m
m
s
d
t
t
t
d
8.5
14.0,8.5
14.0,8.5
6.8
6.8
7.2
7.2
7.2
8.6
Chemical shifts are relative to dimethyl sulfoxide (2.49 ppm).
concentrations of CDP-840 and CT2412 following p.o. or i.v. dosing
were determined by HPLC with UV detection, and were plotted in
Fig. 8. The levels of CDP-840 in rat plasma after 20-mg/kg p.o.
dosing were not detectable. In the i.v.-dosed (5 mg/kg) rats, CDP-840
had a plasma concentration of 4.4 ␮M (average of three rats) at 5-min
postdosing, and no detectable levels of CDP-840 were found after 2 h.
In healthy male volunteers at 16 mg b.i.d dosing with CDP-840, the
half-life of CDP-840 was determined to be 6 h. For CT2412, plasma
levels in p.o.-dosed rats were detected with a Cmax of 0.44 ␮M at 2 h.
In i.v.-dosed rats, a similar concentration (3.7 ␮M) of CT2412 at
5-min postdosing was observed compared with that of CDP-840,
however, the levels of CT2412 could be detected up to 4 h (0.3 ␮M)
postdosing.
In Vivo Metabolism of CDP-840. The plasma samples obtained
after p.o. dosing to rat, rabbit, and human were analyzed. The major
circulating metabolites observed in rat plasma were R4 para-phenol
(M6) and N-oxide (M5). In rabbit plasma, the hydroxy cyclopentyl
(M9) was the major metabolite. In human plasma, pyridinium glucuronide (M12), N-oxide (M5) and hydroxy cyclopentyl (M9) were
detected as major metabolites.
Discussion
FIG. 7. Microsomal metabolism profiles of CT2412 in rat and rhesus monkey by
HPLC/UV (at 245 nm).
Metabolite peaks were assigned by LC/MS, descyclopentyl (1), hydroxy cyclopentyl (2 and 3), N-oxide (4), and desmethyl (5).
N-glucuronide metabolite was not detected for CT2481 in human or
rabbit microsomes supplemented with UDPGA.
The metabolism profiles of CT2412 and CT2481 in rat hepatocytes
were also investigated, and were similar to those in microsomes with
addition of phase II glucuronide conjugates (⫹O⫹gluc, data not
shown). The phase II metabolism, however, was significantly reduced
compared with that of CDP-840 in rat hepatocytes.
In Vivo Pharmacokinetics of CDP-840 and CT2412. Rat plasma
The in vitro metabolism profiles of CDP-840 were evaluated using
hepatic microsomes and freshly isolated hepatocytes from different
species. With the combination of HPLC/UV, LC/MS, NMR, and
available synthetic standards, in vitro phase I and phase II metabolites
of CDP-840 were characterized. Primary CF-LSI mass spectra of
CDP-840 and its metabolites all exhibited abundant molecular ions
and unique fragment ions, allowing the sites of metabolism to be
easily and quickly pinpointed to a particular substructure. NMR data
and synthetic standards provided confirmation of metabolite structures. In our study of CDP-840 metabolism, many synthetic standards
had been or were rapidly synthesized, making some of the metabolite
identification much more straightforward.
The phase I oxidative metabolism profile of CDP-840 was evaluated using microsomal proteins from various species. Up to 10 oxidative metabolites were detected. The reconstructed CF-LSIMS ion
chromatograms of all MH⫹ of metabolites and CDP-840 showed very
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2
5
6
7
8
8⬘
10,13
11,12
15,19
16,18
17
20
21,22,23,24
25
26
27
28
29
30
␦a
IN VITRO METABOLISM STUDIES OF PDE-IV INHIBITORS
239
similar profiles and peak ratios as the analytical HPLC/UV traces. The
pyridyl nitrogen is likely the site for protonation, and therefore the
CF-LSIMS responses for the metabolites and CDP-840 were believed
to be very similar. The sum of the reconstructed ion chromatogram
(Fig. 2) reflected not quantitative but relative amount of metabolites
generated. Several sites of CDP-840 were found to undergo biotransformation. The R1 cyclopentyl group was the major site for metabolism in most species. Several hydroxy cyclopentyl metabolites were
detected. On the R2 catechol ring, both desmethyl and descyclopentyl
metabolites were observed. Similar biotransformations were also reported for rolipram (Krause and Kuhne, 1992, 1993), a PDE-IV
inhibitor from Schering, which has the same catechol moiety. On the
R3 pyridyl, N-oxidation was detected, and on the R4 phenyl, parahydroxylation was observed. The oxidative metabolism of CDP-840
in human microsomes was similar to those in hepatic microsomes
from rhesus monkey, guinea pig, dog, ferret, rabbit, and mouse,
although the relative percentages of each metabolite varied in these
different species. The metabolism profile in rat microsomes, however,
differed significantly from these species. The major site of metabolism in rat involved the para-hydroxylation (M6) on the R4 phenyl
ring. M6 was detected as a minor metabolite in rabbit microsomes,
and was not detected in the microsomal incubations of CDP-840 with
human and other species. The oxidative microsomal metabolism pathways of CDP-840 are summarized in Fig. 9.
Phase II metabolism of CDP-840 was evaluated using hepatocytes.
Freshly isolated hepatocytes retain phase I and phase II enzyme
activities, and therefore, should provide a better correlation with in
vivo metabolism (Placidi et al., 1997; Nicoll-Griffith et al., 1999). Rat
hepatocytes are used as our primary in vitro system for checking
overall metabolic stabilities of synthetic compounds at the early drug
discovery stage, and proved to be very useful to determine the overall
metabolism (both phase I and phase II simultaneously). Because
significant interspecies difference in metabolism were found between
rat and other species from microsomal studies, hepatocytes isolated
from different species were prepared, and their metabolism profiles
were compared. Metabolism profiles of CDP-840 in rat, rabbit, and
human hepatocytes indicated more extensive metabolism relative to
that in microsomes, and provided evidence of additional interspecies
differences in the phase II metabolism. The in vitro phase II metabolism pathways of CDP-840 are summarized in Fig. 10.
In the human hepatocyte incubation of CDP-840, the phase II
metabolism dominated, and the major metabolite was confirmed as R3
FIG. 9. Phase I oxidative microsomal metabolism pathways of CDP-840.
*Major pathways in the species.
pyridinium glucuronide (M12). It was also found to be the major
circulating metabolite of CDP-840 in human plasma. This metabolite
was not detected in the rat hepatocyte incubation. The formation of
pyridinium glucuronides has been reported in the literature for
tripelennamine (Yeh, 1991) and nicotine (Byrd et al., 1992). The food
pyrolysis product 2-amino-1-methy-6-phenylimidazo[4,5-b] pyridine
also forms pyridinium glucuronides in human and rabbit microsomes
under UDPGA, but not in rat microsomes (Styczynski et al., 1993).
The metabolism profile of CDP-840 in rat hepatocytes was very
different from that in human hepatocytes. The extent of metabolism
was also greater in rat compared with that in human or rabbit. Several
metabolites (M6⬘, M13, M16, M17, M18, and M20) were detected in
rat (some were also in rabbit) hepatocytes, but were not present in
human hepatocytes. These metabolites were probably formed through
an epoxide intermediate on the R4 phenyl. The epoxide could isomerize nonenzymatically to the R4 para-phenol (M6). It could also
convert to a R4 dihydrodiol (M13) possibly by epoxide hydrases. The
stable diol could further dehydrate enzymatically to yield the paraphenol (M6), or be oxidized by a dehydrogenase to generate a diphenolic metabolite (M16). The epoxide could also be susceptible to
conjugation (M20) with glutathione by glutathione S-epoxide transferase. The formation of aromatic epoxides and further metabolized
products such as phenols, dihydrodiols, and glutathione conjugates is
well documented in the literature, as in the case of naphthalene (Jerina
et al., 1970) and monohalogenobenzenes (Parke, 1968). The R4
diphenolic metabolite (M16) could undergo O-methylation to form
M6⬘. O-Methylation of catechol metabolites formed by the epoxidedihydrodiol pathway is in the literature for diphenylhydantoin
(Glazko, 1973). The reaction was mediated by catechol O-methyl
transferase (Bakke, 1970). Both M6⬘ and M6 (R4 para-phenol) could
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FIG. 8. Plasma levels of CDP-840 and CT2412 in rats.
240
LI ET AL.
also further conjugate with glucuronic acid to form the corresponding
glucuronides M17 and M18.
In the development of any potential therapeutic drug, the preclinical
safety studies are crucial to evaluate efficacy, pharmacokinetics, and
toxicity. It is very important that the safety species produce the same
metabolites as those found in humans. Rats are typically used as the
early preclinical safety species. The profound interspecies differences
in the in vitro metabolism of CDP-840 between rat and human would
make the preclinical safety studies in the rat invalid. Species differences in metabolism became a critical issue, and one of the important
criteria to select potential backup compounds was to eliminate these
differences. CT2412 was quickly identified, it has similar potency
against PDE-IV (GST-met248A assay: 6.7 nM) compared with that of
CDP-840 (4.3 nM). The para-Cl substitution on R4 eliminated the R4
para-phenol metabolite and other epoxide-mediated metabolites in
rat. This simple substitution improved the metabolic stability in rat,
and most importantly, similar metabolism profiles of CT2412 were
observed in the microsomal incubations with rat and rhesus monkey.
However, in the glucuronidation studies, the N-glucuronide metabolite was still detected in human microsomes and rabbit microsomes at
a rate of 0.1 nmol/min 䡠 mg of protein. This metabolite would
probably be expected in human in vivo, but not in rat. Therefore, it
was important to eliminate this metabolite, and CT2481, which had
N-oxide on R3, was again quickly identified. CT2481 is also equally
potent against PDE-IV (GST-met248A assay: 9.2 nM). The N-oxide
effectively blocked the N-glucuronide metabolite formation in human.
Among several N-oxide compounds we investigated, the reduction of
N-oxide to free pyridyl appeared to be a very minor biotransformation
pathway both in vitro and in vivo; therefore. N-Glucuronidation is
unlikely to occur in vivo with the N-oxide compounds.
Acknowledgments. We acknowledge Dr. Marc Bilodeau from the
Hospital Saint-Luc, Montreal for providing human liver tissue. We
also thank Yves Girard for useful discussions and laboratory animal
resources of Merck Frosst for obtaining the plasma samples from rat
and rabbit.
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