Cross-species comparison of the metabolism and excretion of zoniporide: Contribution of aldehyde oxidase to interspecies differences

Article abstract of DOI:10.1124/dmd.109.030783

Excretion and metabolism of zoniporide were investigated in humans after intravenous infusion of [¹⁴C]zoniporide at an 80-mg dose. Bile was the primary route of excretion because 57% of dose was recovered in the feces after intravenous infusion. Zoniporide was primarily cleared via metabolism in humans. 2-Oxozoniporide (M1) was the major excretory and circulating metabolite in humans and was catalyzed by aldehyde oxidase (K_m of 3.4 μM and V_max of 74 pmol/min/mg protein). Metabolites M2 (17% of the dose) and M3 (6.4% of circulating radioactivity), in which the guanidine moiety was hydrolyzed to a carboxylic acid, were also detected in human feces and plasma, respectively, suggesting that hydrolysis was another route of metabolism of zoniporide in humans. The metabolism and excretion of [¹⁴C]zoniporide in rats and dogs were also evaluated. As in humans, bile was the primary route of excretion of the radiolabeled material in both species, and metabolism was the primary route of clearance. A comparison of plasma metabolites showed that for M3, rats had a higher concentration than human or dog. M1 was absent in dog and present in human and rat plasma at comparable levels, whereas comparison of excreta showed that the total body burden of M1 was greater in rat than that in human. No further evaluation of M2 was considered because it was detected only in the human fecal extracts. Hence, no further toxicological evaluation of the three human metabolites was undertaken. Copyright

Full text of DOI:10.1124/dmd.109.030783

0090-9556/10/3804-641–654$20.00
DRUG METABOLISM AND DISPOSITION
Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
DMD 38:641–654, 2010
Vol. 38, No. 4
30783/3567567
Printed in U.S.A.
Cross-Species Comparison of the Metabolism and Excretion of
Zoniporide: Contribution of Aldehyde Oxidase to
Interspecies Differences
Deepak Dalvie, Chenghong Zhang,1 Weichao Chen,2 Teresa Smolarek, R. Scott Obach, and
Cho-Ming Loi
Pharmacokinetics, Dynamics and Metabolism, Pfizer Global Research and Development, San Diego, California (D.D., C.-M.L.);
and Pharmacokinetics, Dynamics and Metabolism, Pfizer Global Research and Development, Groton, Connecticut (C.Z., W.C.,
T.S., R.S.O.)
Received October 13, 2009; accepted December 29, 2009
ABSTRACT:
Excretion and metabolism of zoniporide were investigated in hu- olism and excretion of [14C]zoniporide in rats and dogs were also
mans after intravenous infusion of [14C]zoniporide at an 80-mg evaluated. As in humans, bile was the primary route of excretion of
dose. Bile was the primary route of excretion because 57% of dose
the radiolabeled material in both species, and metabolism was the
was recovered in the feces after intravenous infusion. Zoniporide primary route of clearance. A comparison of plasma metabolites
was primarily cleared via metabolism in humans. 2-Oxozoniporide showed that for M3, rats had a higher concentration than human or
(M1) was the major excretory and circulating metabolite in humans dog. M1 was absent in dog and present in human and rat plasma at
and was catalyzed by aldehyde oxidase (Km of 3.4 ␮M and Vmax of comparable levels, whereas comparison of excreta showed that
74 pmol/min/mg protein). Metabolites M2 (17% of the dose) and M3
(6.4% of circulating radioactivity), in which the guanidine moiety No further evaluation of M2 was considered because it was de-
was hydrolyzed to a carboxylic acid, were also detected in human tected only in the human fecal extracts. Hence, no further toxico-
the total body burden of M1 was greater in rat than that in human.
feces and plasma, respectively, suggesting that hydrolysis was logical evaluation of the three human metabolites was undertaken.
another route of metabolism of zoniporide in humans. The metab-
Zoniporide [1-(quinolin-5-yl)-5-cyclopropyl-1H-pyrazole-4-carbonyl gua- evaluated. There was a linear increase in systemic exposure of zo-
nidine] (Fig. 1) was designed and synthesized as a highly selective
inhibitor of the sodium/hydrogen exchanger (NHE-1) (Guzman-Perez
et al., 2001; Knight et al., 2001; Marala et al., 2002; Tracey et al.,
2003) and was being developed to reduce the perioperative myocar-
dial ischemic injury in high-risk surgery patients. It inhibited 22Naϩ
uptake in fibroblasts expressing human NHE-1 in a concentration-
dependent manner with an IC50 of 14 nM and had Ͼ150-fold selec-
tivity over other NHE isoforms (Marala et al., 2002). Zoniporide also
produced a reduction in infarct size and decreased the incidence and
duration of potentially lethal arrhythmias after intravenous adminis-
tration to animal models of ischemic-reperfusion injury (Knight et al.,
2001; Tracey et al., 2003).
niporide in healthy male human volunteers, after a 1-h infusion of
doses ranging from 0.003 to 1.0 mg/kg, as assessed by Cmax and the
area under plasma concentration versus time curve (AUC0–tlast). The
Cmax and AUC0–tlast ranged from 2 to 622 ng/ml and from 2 to 930
ng-h/ml, respectively, at these doses. The Clp and Vd were indepen-
dent of dose, and the mean t1/2 was approximately 3 h. The most
common adverse events reported by subjects receiving zoniporide
were somnolence and postural hypotension. Other adverse events
included nausea, vomiting, and headache.
In drug development, it is important to study the metabolism and
the routes of excretion of a drug candidate in humans. Furthermore,
assurance that all major circulating metabolites observed in humans
are present in at least one animal species used for safety assessment
provides greater confidence that animal toxicology studies are rele-
vant for human safety (Baillie et al., 2002; Smith and Obach, 2005,
2006; Davis-Bruno and Atrakchi, 2006; Atrakchi, 2009). The recent
Food and Drug Administration (2008) guidance on metabolites in
safety testing recommends that human circulating metabolites exceed-
ing 10% of the parent should be present in at least equal quantities in
at least one of the preclinical species used in toxicological assessment
(Davis-Bruno and Atrakchi 2006; Atrakchi 2009). Direct testing in
Preclinical pharmacokinetic studies of zoniporide in mice, rats,
rabbits, dogs, and monkeys demonstrated that its clearance (Clp) was
moderate to high in all species. The volume of distribution (Vd) was
moderate, and the plasma t1/2 was 1.5 h or shorter in all species
1 Current affiliation: Genentech, San Francisco, California.
2 Current affiliation: Arena Pharmaceuticals, San Diego California.
Article, publication date, and citation information can be found at
http://dmd.aspetjournals.org.
doi:10.1124/dmd.109.030783.
ABBREVIATIONS: NHE-1, sodium/hydrogen exchanger; CL, total clearance; AUC, area under plasma concentration versus time curve; SBE,
sulfobutylether; HPLC, high-performance liquid chromatography; MS, mass spectrometry; RAM, radioactivity monitoring detector; LC-ARC, liquid
chromatography-accurate radioisotope counting system; CID, collision-induced dissociation; amu, atomic mass units; P450, cytochrome P450.
641

642
DALVIE ET AL.
All volunteers were administered a single 80-mg dose of [14C]zoniporide
HN
mesylate (100 ␮Ci) by intravenous infusion over 1 h in water at a 10 mg/ml
concentration. The specific activity of the dose was 1.25 ␮Ci/mg. Urine was
collected into containers surrounded by dry ice at predose (Ϫ8 to 0), 0 to 12,
and 12 to 24 h and at 24-h intervals during the study through 144 h postdose
(8 days). Feces were collected before dosing and then over 24-h intervals up
to 144 h postdose (8 days). The total weight of the urine and feces was
recorded after each collection. Blood samples (sufficient to provide 6 ml of
plasma) were collected for pharmacokinetic evaluation of total radioactivity
and zoniporide at times 0 (just before dosing) and at 0.5, 1, 2, 4, 8, 12, 24, 36,
48, and 24 h after the start of the infusion. Subsequent samples were taken at
24-h intervals through 144 h. Additional blood sufficient to provide a mini-
mum of 20 ml of plasma was collected at 1, 4, 8, and 12 h postdose from the
start of the infusion for characterization of metabolites. All samples were
collected in heparinized tubes and centrifuged at approximately 4°C. Samples
were stored frozen until the day of analysis.
O
NH2
NH
*
N
N
Animal Studies. All studies were conducted in a research facility accredited
by the American Association for the Accreditation of Laboratory Animal Care.
All study animals were acclimated to standard housing and environmental
conditions in metabolism cages and rooms where light cycles, temperature, and
humidity were documented daily for 2 days before the experiments.
N
FIG. 1. Structure of [14C]zoniporide. ء, position of the radiolabel.
Rats. Three male and three female Sprague-Dawley rats were housed
individually in Nalgene metabolism cages and fasted overnight. A single dose
of [14C]zoniporide was administered to all animals intravenously at a dose of
10 mg/kg containing 50 ␮Ci of radioactivity. The dose was prepared by
dissolving [14C]zoniporide in 20% SBE-␤-cyclodextrin at a concentration of
2.1 mg/ml. The specific activity of the dose was 28.8 ␮Ci/mg. Urine was
collected into containers surrounded by dry ice at predose (Ϫ24 to 0), 0 to 8,
and 8 to 24 h and at 24-h intervals during the study and feces over 24-h
intervals, up to 168 h postdose. After the last urine and fecal collection, the
cages were washed with 1:1 ethanol-water (v/v), and the wash was collected.
The weight of each fecal sample, cage wash, and cage debris was determined.
All animals were euthanized by CO2 asphyxiation at the end of the study. For
pharmacokinetic studies, six Sprague-Dawley rats (n ϭ 3/sex) were dosed
intravenously with 10 mg/kg [14C]zoniporide. The dose was prepared by
mixing unlabeled and radiolabeled zoniporide in 20% w/v SBE-␤-cyclodex-
trin. The concentration and specific activity of the dose was 3.34 mg/ml and
7.46 ␮Ci/mg, respectively. Blood samples were collected in heparinized tubes
at times 0 (just before dosing) 3, 15, 30, 60, 120, 240, 360, and 480 min. After
sampling, whole blood was centrifuged at 14,000 rpm for 3 min, and plasma
was transferred to Eppendorf snap-capped tubes and stored at Ϫ20°C until
assay. For identification of circulating metabolites, a separate group (two
animals, one per sex, per each time point) of animals were dosed intravenously
at a dose of 10 mg/kg [14C]zoniporide (containing 75 ␮Ci of radioactivity).
The specific activity of the dose was 30 ␮Ci/mg. Blood was collected at 1, 2,
4, 8, and 24 h by sacrificing the rats at each sampling time. Samples were
collected in heparinized tubes and centrifuged at approximately 4°C. All
samples were stored frozen at Ϫ20°C until the day of analysis.
animals is warranted for human metabolites that are absent or present
in disproportionately lower amounts in preclinical species used for
toxicology studies.
Conventionally, an assessment of major metabolites in humans and
preclinical species is accomplished by definitive absorption, distribu-
tion, metabolism, and excretion studies in which radiolabeled drugs
are administered and biological fluids are evaluated for a comprehen-
sive and quantitative profile of metabolites. The current study was
performed to compare the metabolism and excretion of zoniporide in
humans and animal species used in toxicological testing using radio-
labeled zoniporide. [14C]Zoniporide (Fig. 1) was administered to four
young healthy male volunteers by intravenous infusion for 1 h, and the
excreta and plasma were collected to assess the mass balance, routes
of excretion, and circulating metabolites of zoniporide in humans. To
assess the coverage of all human metabolites in toxicology species,
[14C]zoniporide was also administered to Sprague-Dawley rats and
Beagle dogs, and the metabolites in the excreta and in circulation were
also identified to determine the major metabolic routes of zoniporide.
In addition, the characterization of the enzymes responsible for the
formation of M1 in humans and the enzyme kinetics for the formation
of M1 were also evaluated in vitro in this study using human subcel-
lular fractions.
Materials and Methods
Dogs. Four noncannulated (two per gender) Beagle dogs were housed
individually in stainless steel metabolism cages and administered [14C]zonipo-
ride by intravenous administration at a dose of 3 mg/kg (containing 150 ␮Ci
of radioactivity). The dose was prepared by dissolving the salt of unlabeled and
labeled zoniporide (specific activity 26.6 mCi/mmol) into 20 ml of 20%
SBE-␤-cyclodextrin. The specific activity of the dose was 5 ␮Ci/mg. Urine
was collected in containers surrounded by dry ice at predose (Ϫ12 to 0), 0 to
8, and 8 to 24 h and at 24-h intervals during the study through 168 h postdose.
Feces were collected before dosing and then over 24-h intervals, up to 168 h
postdose. The total weight of urine and feces was recorded after each collec-
tion. Blood samples were collected for pharmacokinetic evaluation of zonipo-
ride and total radioactivity at times 0 (just before dosing) 0.5, 1, 2, 4, 8, 12, and
24 and at 24-h intervals through 168 h postdose. For characterization of
circulating metabolites, additional blood was collected at 0.5, 1, 2, 4, 8, and
Reference Compounds, Radiolabel Zoniporide, and Chemicals. All syn-
thetic standards for the metabolites were synthesized at Pfizer Global Research
and Development (Groton, CT) using standard procedures. [14C]Zoniporide
mesylate was synthesized by the radiochemistry group at Pfizer Global Re-
search in Groton under good manufacturing practice conditions. The label was
located in the pyrazole ring of the molecule (Fig. 1). The purity of the
radiolabeled material was Ͼ99%. For animal studies, [14C]zoniporide (radio-
chemical purity 99.4%, specific activity was 26.6 mCi/mmol) was synthesized
by the radiochemistry group at Pfizer Global Research in Groton. All other
reagents and solvents used in the studies were of the highest grade available
and were obtained from commercial sources. EcoLite(ϩ) scintillation cocktail
was obtained from ICN Pharmaceuticals (Irvine, CA). Carbo-Sorb and Per-
mafluor Eϩ scintillation cocktails were purchased from PerkinElmer Life and
Analytical Sciences (Waltham, MA).
Study Design, Dosing, and Sample Collection in Humans. This was an 12 h after an intravenous dose. Samples were collected in heparinized tubes
open-label, single-dose inpatient study conducted with four nonsmoking
healthy male human volunteers aged between 18 and 55 years and weighing
between 74 and 79 kg. Before the study started, an institutional review
committee approved the protocol and the informed consent document. All
study participants gave written informed consent before initiation of the study.
and centrifuged at approximately 4°C. All samples were stored frozen at
Ϫ20°C until the day of analysis.
Quantitation of Radioactivity. Radioactivity in the plasma, urine, and
feces was determined by liquid scintillation counting. Aliquots of plasma
(50–100 ␮l) and urine (100–500 ␮l) were mixed with EcoLite(ϩ) scintillation

METABOLISM AND EXCRETION OF ZONIPORIDE
643
cocktail (6 ml) and counted in triplicate in a model L 1409 DSA liquid
more to ensure that Ͼ90% of the radioactivity was recovered. The supernatants
scintillation counter (PerkinElmer Life and Analytical Sciences–Wallac Oy, were mixed and evaporated to dryness in a TurboVap at 35°C under nitrogen.
Turku, Finland). For determination of radioactivity in feces, the weight of each
fecal sample was determined, and the samples were homogenized in 2 parts of
The residues were reconstituted in 100 ␮l of 5 mM ammonium formate (pH 3),
and an aliquot (100 ␮l) was injected onto the column. Aliquots (30 ␮l) of the
deionized water using a Stomacher blender 400. After homogenization, trip- reconstituted samples were also counted on the liquid scintillation counter to
licate aliquots (250–500 ␮l) of each sample were transferred into tared cones determine the radioactivity extraction recovery.
and pads, weighed, and combusted in an automatic sample PerkinElmer 308
Feces. Fecal homogenates were pooled on a weight basis to account for 90%
oxidizer (PerkinElmer Life and Analytical Sciences). The resulting 14CO2 was or greater of the drug-related material excreted in feces. Each pooled fecal
trapped in Carbo-Sorb and mixed with Permafluor Eϩ scintillation fluid, and sample was diluted with 30 ml of acetonitrile and vortexed. The sample was
the radioactivity was quantified by liquid scintillation counting. Radioactivity
less than twice the background value was considered to be below the limit of
then centrifuged, and the supernatant was separated. The process was repeated
several times until Ͼ90% of the radioactivity was extracted. All supernatants
determination. Samples collected before dosing were used as controls and were mixed and evaporated to approximately 1 ml in a TurboVap at 35°C
counted to obtain a background count rate.
under nitrogen. The concentrated residue was extracted with 10 ml of hexane
to remove all lipophilic material, and the aqueous layer was evaporated to
dryness in a TurboVap at 35°C under nitrogen. The residue obtained was
reconstituted in ϳ300 ␮l of 3 mM ammonium formate solution, and a small
The radioactivity in the plasma was expressed as nanogram-equivalents of
zoniporide per milliliter. The compound equivalents were determined by
dividing the microcuries per milligram of sample by the specific activity of the
compound (1.25 ␮Ci/mg). Samples containing radioactivity (disintegrations sample was analyzed by liquid scintillation counting for radioactivity extrac-
per minute) less than or equal to twice the background were considered to be
zero in the calculation of the means. Radioactivity in the urine and feces was
expressed as a percentage of the administered dose per time interval.
tion recovery. A 100-␮l aliquot of the reconstituted sample was injected onto
the column.
Separation, Quantification, and Identification of Metabolites. Metabolic
Quantitation of Zoniporide in Human, Rat, and Dog Plasma. Plasma profiling was performed using the HPLC system that consisted of an HP-1100
samples were analyzed for zoniporide using a validated liquid chromatogra-
phy-tandem mass spectrometry method. A 200-␮l aliquot of each human
plasma sample or a 50-␮l sample from rat and dog plasma was treated with 100
membrane degasser, HP-1100 autoinjector, and HP-1100 binary gradient pump
(Agilent Technologies, Palo Alto, CA). Chromatography was performed on a
Zorbax C8 column (5 ␮m, 4.5 ϫ 150 mm) by injecting 100 ␮l of the
␮l of a solution of an internal standard (100 ␮g/ml [15N3]zoniporide). The reconstituted sample. The mobile phase was initially composed of acetonitrile
samples were basified with sodium carbonate solution, and the analytes were
extracted with methyl tert-butyl ether. The organic layer was separated and
(solvent B) and 10 mM ammonium formate (pH 3.0) (solvent A). The flow rate
was 1.0 ml/min, and separation was achieved at ambient temperature. A
evaporated to dryness, and the residue was reconstituted with 200 ␮l of a 50-min gradient was used as follows: 0 to 10 min, 5% B; 10 to 20 min, 10%
mixture of acetonitrile and 2 mM ammonium acetate (15:85), the extracts were
analyzed using a PE Sciex API 3000 mass spectrometer with a TurboIonSpray
source (PerkinElmerSciex Instruments, Waltham, MA). The analytes were
B; 20 to 35 min, 20% B; 35 to 40 min, 20% B; 40 to 44 min, 80% B; and 44
to 45 min, 5% B. The column was reequilibrated at 5% B for the next 5 min
before next injection. The postcolumn eluate was split such that 95% of the
separated chromatographically using a Betabasic C18 column (100 ϫ 2 mm, 5 flow was monitored continuously with a RAM fitted with a liquid scintillation
␮m) and detected in a positive ion mode using the protonated molecular ion as cell (IN/US Systems, Riviera Beach, FL). The remaining 5% of the flow was
the precursor ion monitored at m/z 321.0 Ͼ 262.0 for zoniporide and at m/z diverted to the PE Sciex API 3000 triple quadrupole mass spectrometer. The
324.0 Ͼ 262.0 for the internal standard. Data collection and integration were RAM was operated in the homogeneous liquid scintillation counting mode
performed using Sample Control and MacQuan version 1.5 software (MDS with the addition of 3 ml/min of Tru-Count scintillation cocktail (IN/US
Sciex, Concord, ON, Canada). Quantitation was based on a linear least-squares Systems) to the effluent. The RAM response was recorded as a real-time
regression analysis of calibration curves weighted 1/x2 using the area ratio analog signal by the MS data collection system.
versus concentration. The dynamic range of the assay was 1.00 to 500 ng/ml
for both zoniporide and M1.
The metabolites in the urine and feces were quantified by measuring
radioactivity in the individually separated peaks in the radiochromatogram
Determination of the Pharmacokinetic Parameters. Pharmacokinetic using Winflow software (IN/US Systems). The RAM provided an integrated
parameters were determined by noncompartmental methods from individual con- printout in counts per minute and percentage of the radiolabeled material. The
centration time profiles after intravenous infusion using WinNonlin (version 3.2; circulating metabolites were detected by an LC-ARC (AIM Research, Hockes-
Pharsight, Mountain View, CA). The maximum plasma concentration (Cmax) and sen, DE). The LC-ARC was operated in the homogeneous liquid scintillation
the time at which this concentration was achieved (Tmax, at the end of the infusion) counting mode with the addition of 2.5 ml/min of Tru-Count scintillation
were taken directly from the concentration data. The AUC0–tlast was calculated cocktail (IN/US Systems) to the effluent. The circulating metabolites were
from 0 to the last quantifiable time point (tlast), using log-linear trapezoidal quantified using the LC-ARC software (AIM Research) by measuring the
approximation. The plasma terminal elimination rate constant (kel) was estimated radioactivity in the chromatographically separated peaks.
by linear regression analysis of the terminal slope of log plasma concentration-time
The metabolites were identified using a PE Sciex API 3000 mass spectrom-
curve. The terminal elimination half-life (t1/2) was estimated as ln2/kel. The total eter equipped with an electrospray ion source operated in the positive ion
clearance (CL) was determined by the ratio CL ϭ dose/AUC0–ϱ. The area under mode. The instrument settings and potentials (e.g., collision energy) were
the plasma concentration-time curve from zero to ϱ (AUC0–ϱ) was estimated as adjusted to provide optimal data for zoniporide. The PE Sciex API 3000 mass
the sum of AUC0–tlast and AUCtlast–ϱ. The AUCtlast–ϱ was estimated from Ctlast/kel, spectrometer was operated at an ionspray voltage of 4000 V and orifice voltage
where Ctlast represented the estimated plasma concentration at Tlast, based on the of 36 V. The collision-induced dissociation (CID) studies (precursor ion scan
aforementioned regression analysis. The volume of distribution (Vss) was deter- or product ion scan) were performed using nitrogen gas at a collision energy
mined by CL ⅐ MRT, with MRT being the mean residence time after intravenous of 30 V and the collision gas thickness of 4 ϫ 1014 molecules/cm2. The MS
infusion.
data were analyzed by MultiView 1.4 software (PerkinElmerSciex Instru-
Metabolic Profiling. Urine. Urine was pooled from 0 to 24 h so that Ͼ90% ments). The metabolites were identified using Q1 (full scan), neutral loss, and
of the drug-related material excreted in urine was accounted for. The pooled precursor ion-scanning techniques. The structures of metabolites were identi-
urine samples were lyophilized overnight, and the residue was dissolved in 1
ml of water. The pooling was proportional to the volume of urine collected at
each time point. The aqueous layer was centrifuged, and an aliquot (100 ␮l)
was injected onto a column.
fied using a product ion scan of the molecular ions that were identified in the
above scanning modes and multiple reaction monitoring scanning.
In Vitro Metabolism of Zoniporide. Zoniporide (10 ␮M) was incubated
with human liver S9 fractions (protein concentration 2.5 mg/ml), MgCl2 (3.3
Plasma. Plasma samples obtained were pooled to account for Ͼ90% of the mM), in the presence of NADPH (3.0 mM) in a total volume of 1.0 ml of
radioactivity using the method reported by Hamilton et al. (1981) for profiling potassium phosphate (0.1 M, pH 7.4). Incubations were started by addition of
of circulating metabolites. The pooled samples were treated with 5 parts of NADPH and shaken in a water bath set at 37°C. Control experiments were
acetonitrile to 1 part of plasma. The mixture was then centrifuged, and the carried out in a similar manner except that the NADPH solution was substi-
supernatant was transferred to another tube. The pellets were washed once tuted with phosphate buffer. After 1 h, the incubations were quenched with

644
DALVIE ET AL.
acetonitrile (5 ml) and centrifuged, and the supernatant was evaporated to
dryness in a TurboVap under nitrogen. The residue was reconstituted in 200 ␮l
of acetonitrile and water mixture (1:3), and a 25-␮l aliquot was injected onto
the column for the identification of the oxidation products. Enzyme phenotying
studies with zoniporide were carried out in a similar manner except that the
incubation mixture was first treated with raloxifene (10 ␮M), an inhibitor of
aldehyde oxidase, before addition of the substrate.
The metabolites in the in vitro samples were identified by a Finnigan LCQ
Deca XP ion trap mass spectrometer (Thermo Fisher Scientific, Waltham, MA)
equipped with an electrospray ion source. The mass spectrometer was operated
in a positive ion mode, and the operating parameters for the ion trap were as
follows: capillary temperature, 270°C; spray voltage, 4.0 kV; capillary voltage,
4.0 V; sheath gas flow rate, 90; and auxiliary gas flow rate, 30. The mass
spectrometer was operated in a positive ion mode with data-dependent scan-
ning. The ions were monitored over a full mass range of m/z 125 to 1000. For
Results
Excretion Studies. Healthy human volunteers. Urine and feces were
collected over 144 h (6 days) from four healthy male volunteers after
intravenous infusion of 80 mg of [14C]zoniporide for 1 h. A mean 91%
of the dose was recovered in the urine and feces over 144 h (Table 1). The
majority of dose (Ͼ95%) was excreted within the first 96 h after admin-
istration of the radioactive dose in all subjects (Fig. 2A). Bile was the
major route of excretion because 57% of the dose was excreted in the
feces after an intravenous infusion of [14C]zoniporide (Table 1). Approx-
imately 34% of the total dose was excreted in the urine (Table 1).
Rats. A mean 89% of the dose was recovered after a single bolus 10
mg/kg dose of [14C]zoniporide to Sprague-Dawley rats (Table 1). Most
of the radioactivity was recovered in the initial 48 h in all rats (Fig. 2B).
data-dependent scanning, the normalized collision energy was 40%. The MS As in humans, the majority of the dose (61%) was excreted in the feces
data were analyzed using Xcalibur software (version 1.4; Thermo Fisher
Scientific).
of rats and only 28% of the dose was excreted in the urine after 168 h.
This result suggested that bile was the primary route of excretion of
zoniporide material in rats as well.
Dogs. Approximately 89% of the dose was recovered in the urine and
feces of dogs 168 h after intravenous administration of [14C]zoniporide at
a dose of 3 mg/kg. Bile was the primary route of excretion in dogs as well
because 62% of the administered dose was excreted in the feces of this
species, whereas 27% of the dose was recovered in the urine of dogs
Enzyme Kinetics. Zoniporide (1–500 ␮M) was incubated with pooled
human liver cytosol (0.2 mg/ml; Sigma-Aldrich, St. Louis, MO) and EDTA
(0.1 mM) in 0.2 ml of 25 mM phosphate buffer at 37°C for 60 min. (Prelim-
inary experiments demonstrated that the protein concentration and incubation
time used provided linear reaction velocity.) The incubations were commenced
with addition of cytosol and terminated with the addition of 0.05 ml of 1 M
formic acid. The terminated incubation mixtures were filtered through a
Millipore Multiscreen-HA filter plate (0.45 ␮m), and filtrates were analyzed (Table 1). As in rats most of the dose was recovered in the first 48 h after
by HPLC-MS. Filtered incubation mixtures were injected (0.04 ml) onto a
Luna C18 column (2.5 ϫ 50 mm, 5 ␮; Phenomenex, Torrance, CA) preequili-
brated in 0.1% HCOOH containing 5% CH3CN at a flow rate of 0.4 ml/min.
The HPLC system consisted of two Shimadzu LC-10AD pumps (Shimadzu,
Columbia, MD), a CTC PAL autoinjector (CTC Analytics, Carrboro, NC), and
a Micromass Ultima tandem quadrupole mass spectrometer (Micromass, Bev-
erly, MA) operated in the multiple reaction monitoring mode. The mobile
phase was maintained at initial conditions for 0.5 min followed by a linear
gradient to 70% CH3CN at 3 min. The effluent was introduced into an ionspray
source in positive mode, and tune file parameters were as follows: capillary,
3.5; cone, 25; source temperature, 135°C; desolvation temperature, 350°C;
cone gas, Ϸ190; desolvation gas, Ϸ750; entrance, Ϫ5; collision, 20; and exit,
1, with other potentials optimized to maximize the signal. Metabolite M1 (Rt ϭ
1.98 min) was monitored with the transition m/z 337 to 278. Quantitation was
accomplished by extrapolation from a standard curve ranging from 0.1 to 10
␮M. Assay standards were within Ϯ9% of nominal values.
dosing of the radiolabeled compound (Fig. 2C).
Pharmacokinetics of Total Radioactivity and Zoniporide. Phar-
macokinetics in healthy human volunteers. The mean plasma concen-
tration versus time profiles of total radioactivity and zoniporide after
intravenous infusion of [14C]zoniporide in healthy male volunteers are
presented in Fig. 3A. The Cmax of zoniporide (590 ng/ml) was approxi-
mately 66% of the Cmax of total radioactivity (899 ng-Eq/ml) and oc-
curred at the end of infusion (1 h) of [14C]zoniporide (Table 2). The
AUC0–tlast of zoniporide (833 ng-h/ml) accounted for ϳ28% that of
the AUC(0-tlast) of total circulating radioactivity (2925 ng-Eq-h/ml). The
half-life of total radioactive material was 6.7 h. Unchanged zoniporide
TABLE 1
Percent recovery of the dose in healthy male volunteers, Sprague-Dawley rats,
and Beagle dogs after intravenous administration of [14C]zoniporide
Because the initial velocity versus the substrate concentration plot showed
a decrease in the rates at higher substrate concentrations, the enzyme kinetic
data were fit to the uncompetitive substrate inhibition model using the enzyme
kinetics module of SigmaPlot (version 9.01; Systat Software, Inc., San Jose,
CA) (eq. 1):
͓
14C͔Zoniporide was administered intravenously to humans at a dose of 80 mg and to
Sprague-Dawley rats and Beagle dogs at a dose of 10 and 3 mg/kg, respectively.
% of Dose
Time
Urine
Feces
Total
Healthy male volunteers
0–24 h
0–48 h
0–72 h
0–96 h
0–120 h
0–144 h
Rats
0–24 h
0–48 h
0–72 h
0–96 h
0–120 h
0–144 h
0–168 h
Dogs
0–24 h
0–48 h
0–72 h
0–96 h
Vmax ⅐[S]
v ϭ
(1)
33
34
34
34
34
34
2.3
33
56
56
56
35.3
67
90
90
90
[S]
Km ϩ [S] ϩ
ͩ ͪ
Ksi
in which Vmax and Km are the terms for the standard Michaelis-Menten
parameters maximum reaction velocity and substrate concentration at half-
maximum velocity and Ksi is the constant describing the substrate inhibition
interaction (Houston and Kenworthy, 2000). The intrinsic clearance value was
then determined from the Vmax and Km using eq. 2:
57
91
27
28
28
28
28
28
28
57
59
60
60
60
61
61
84
86
88
88
88
89
89
Clint ϭ Vmax/Km
(2)
The IC50 value was determined from eq. 3:
25
26
26
27
27
27
27
47
58
60
61
61
61
62
71
84
86
88
88
88
89
B⅐[I]
% of control activity ϭ 100⅐ A Ϫ
(3)
ͩ
ͪ
[I] ϩ IC50
0–120 h
0–144 h
0–168 h
in which the terms A and B represent maximum and minimum control
activities at [I] ϭ 0 and [I] ϭ ϱ, respectively.

METABOLISM AND EXCRETION OF ZONIPORIDE
645
A
B
100
100
80
60
40
20
0
80
Urine
Feces
To t a l
60
Urine
FIG. 2. Mean urine and fecal cumulative
recovery of total radioactivity versus time
profile in healthy male volunteers (n ϭ 4)
(A), Sprague-Dawley rats (n ϭ 6) (B), and
Beagle dogs (n ϭ 4) (C) after intravenous
administration of [14C]zoniporide. The er-
rors bars indicate S.D. Healthy male volun-
teers were administered [14C]zoniporide by
intravenous infusion for 1 h at a dose of
80 mg (100 ␮Ci), and the excreta were
collected over 144 h. The rats were ad-
ministered a bolus of [14C]zoniporide at a
dose of 10 mg/kg (50 ␮Ci), and the ex-
creta were collected over 168 h. The dogs
were administered a bolus of [14C]zo-
niporide at a dose of 3 mg/kg (150 ␮Ci),
and the excreta were collected over
168 h.
Feces
40
Tot al
20
0
0
50
100
150
200
0
50
100
150
Time (h)
Time (h)
C
100
80
60
40
20
0
Urine
Feces
To t a l
0
50
100
Time (h)
150
200
A
10000
1000
100
10
Total Radioactivity (ng-equiv/mL)
Zoniporide (ng/mL)
1
0
5
10
15
20
25
30
Time (h after start of infusion)
B
C
10000
10000
1000
100
10
Total Radioactivity (ng-equiv/mL)
Zoniporide (ng/mL)
Total Radioactivity (ng-equiv/mL)
Zoniporide (ng/mL)
1000
100
10
1
1
0
2
4
6
8
0
2
4
6
8
10
12
14
Time (h)
Time (h)
FIG. 3. Mean plasma concentration-time profiles for total radioactivity and zoniporide after intravenous administration of [14C]zoniporide to healthy male volunteers (n ϭ
4) (A), Sprague-Dawley rats (n ϭ 6) (B), and Beagle dogs (n ϭ 4) (C). The errors bars indicate S.D. Healthy male volunteers were administered [14C]zoniporide by
intravenous infusion for 1 h at a dose of 80 mg (100 ␮Ci). The rats were administered a bolus of [14C]zoniporide at a dose of 10 mg/kg (50 ␮Ci). The dogs were administered
a bolus of [14C]zoniporide at a dose of 3 mg/kg (150 ␮Ci).
was rapidly cleared in humans after intravenous infusion of [14C]zonipo- (1390 ng-h/ml) accounted for 39% of AUC0–tlast of total radioactivity
ride with a clearance of 21 ml/min/kg and a half-life of 2.0 h (Table 2). (3570 ng-Eq-h/ml) in rats (Table 2). The mean terminal elimination
Rat. Figure 3B depicts the mean plasma concentration versus time half-life of total circulating radioactivity was 2.0 h and was 5-fold longer
profile for total radioactivity, unchanged zoniporide after a single bolus than that of zoniporide.
intravenous administration of [14C]zoniporide to rat. As in humans,
Dog. After a bolus intravenous administration of 3 mg/kg [14C]zo-
zoniporide was rapidly cleared from rats (Cl ϭ 113 ml/min/kg) with a niporide, the mean AUC0–tlast of total radioactivity and unchanged zo-
half-life of 0.4 h (Table 2). The AUC0–tlast of unchanged zoniporide niporide were 3160 ng-Eq-h/ml and 2009 ng-h/ml, respectively (Table 2).

646
DALVIE ET AL.
TABLE 2
Mean pharmacokinetic parameters of total circulating radioactivity and zoniporide in healthy male volunteers, Sprague-Dawley rats, and Beagle dogs after
intravenous administration of [14C]zoniporide
Healthy male volunteers were administered ͓14C͔zoniporide by intravenous infusion for 1 h at a dose of 80 mg (100 ␮Ci). The Sprague-Dawley rats and Beagle dogs were administered a
bolus of ͓14C͔zoniporide at a dose of 10 mg/kg (50 ␮Ci) and 3 mg/kg (150 ␮Ci), respectively.
Healthy Male Volunteers
Radioactivity* Zoniporide
Mean S.D. Mean S.D.
Sprague-Dawley Rats
Radioactivity* Zoniporide
Mean S.D. Mean S.D.
Beagle Dogs
Pharmacokinetic Parameters
Radioactivity*
Mean S.D.
Zoniporide
Mean S.D.
Tmax (h)
1.0
899
2925
3250
0.0
237
223
122
1.0
590
833
839
0.0
44.2
33.4
34.2
0.4
Cmax (ng/ml)
AUC0–tlast (ng-h/ml)
AUC0–ϱ (ng-h/ml)
t1/2 (h)
3570
3650
323
307
0.83
1390
1490
0.4
113
2.2
287
273
0.10
22
0.58
3160
3330
672
673
0.34
2009
2370
2.51
21
3.7
582
683
0.59
4.7
0.59
6.7
3.3
2.0
2.00
2.80
Cl (ml/min/kg)
Vss (l/kg)
21.0
1.7
1.2
0.4
* The radioactivity was expressed as ng-Eq/ml.
A
The plasma concentration versus time profile for the total radioactivity
and unchanged zoniporide is shown in Fig. 3C. Thus, in contrast to rats
and humans, the exposure of unchanged zoniporide accounted for 64% of
circulating total radioactivity in dogs. Furthermore, in contrast to rats and
humans, the clearance of zoniporide was moderate (21 ml/min/kg),
whereas the Vdss was higher (3.7 l/kg) in dogs. The mean terminal
elimination half-lives of total circulating radioactivity and unchanged
zoniporide were similar (2.8 and 2.5 h, respectively).
2000
1600
1200
800
Zoniporide
M1
400
Metabolite Profiles of Zoniporide in the Urine and Feces.
Healthy male volunteers. Only two metabolites (M1 and M2) were
detected in the urine and feces of humans (Fig. 4). Metabolite M1 was
the primary metabolite detected in the excreta (52% of the total dose),
whereas zoniporide and metabolite M2 represented 18 and 17% of the
total dose (Table 3).
Rat. Representative HPLC radiochromatograms of the extracted uri-
nary and fecal (pooled from 0 to 24 h) samples from rat are shown in
Fig. 5. As in humans, M1 was the primary excretory metabolite in rat and
represented 35% of the total dose (Table 3). Unchanged zoniporide also
constituted the majority of the dose in the urine and feces and accounted
for 40% of the dose. The other metabolites M3 to M7 were minor
metabolites and represented 0.8 to 5.9% of the dose (Table 3).
Dog. Unlike the rat and humans, unchanged zoniporide was the
primary constituent in the excreta of dogs (Fig. 6) and accounted for 37%
of the total dose (Table 3). Major metabolites were M8 and M10, which
accounted for 13 and 18% of the total dose, respectively. Other minor
0
10
20
30
40
50
Time (min)
B
2000
1600
1200
800
M1
Zoniporide
M2
400
0
10
20
30
50
40
Time (min)
FIG. 4. Representative HPLC radiochromatogram of pooled urine (A) and feces (B)
after intravenous infusion of [14C]zoniporide for 1 h at a dose of 80 mg (100 ␮Ci)
to healthy male volunteers.
metabolites M3, M5, M7, and M9 detected in the excreta of the dog plasma, zoniporide and M1 constituted 36 and 46% of the circulating
accounted for 8.6, 5.4, 4.1, and 2.1% of the total dose, respectively (Table radioactivity, whereas the other metabolites, M3 and M6 constituted 9.0
3). Most of these metabolites were excreted in the feces, suggesting and 8.5% of the circulating radioactivity, respectively (Table 4).
biliary excretion of the radiolabeled material.
Dog. The HPLC radiochromatogram of circulating metabolites in
Circulating Metabolites of Zoniporide. Human circulating me- plasma pooled from 1 to 12 h is shown in Fig. 7C. At least 99% of the
tabolites. Circulating metabolites in humans were profiled by pooling total circulating radioactivity in the pooled plasma was identified. Four
the plasma samples obtained over 0 to 12 h using the method described radioactive peaks including zoniporide were observed in dog plasma.
by Hamilton et al. (1981) (Fig. 7A). This method gave a good assessment Zoniporide was the major component circulating and accounted for 62%
of the overall exposure of each metabolite over 0 to 12 h. Approximately of the circulating radioactivity (Table 4). The major circulating metabo-
99% of the total circulating radioactivity could be identified. Three lite was M9 and represented 23% of the total circulating radioactivity,
radioactive peaks were observed in plasma and corresponded to M1, whereas other metabolites (M3, M4, M8, and M10) accounted for 2.3 to
zoniporide, and metabolite M3 (Fig. 7A). Metabolite M1 was the primary 5.1% of the circulating radioactivity (Table 4).
circulating metabolite and accounted for 60% of the circulating radioac-
tivity, whereas zoniporide and M3 accounted for 30 and 6.4% of the total ing Metabolites in Toxicology Species. Both metabolites M1 and M3
circulating radioactivity (Table 4). detected in the plasma of humans were Ͼ10% of the parent as measured
Assessment and Comparison of Exposures of Human Circulat-
Rat. Circulating metabolites in the rat were identified after the plasma by the ratio of the percentage of radioactivity of the metabolite to the
samples obtained from 0 to 24 h were pooled according to the Hamilton percentage of radioactivity of the parent drug as observed in the human
pooling method. All circulating radioactivity in the rat plasma was circulating metabolic profile. Therefore, in accordance with the guidance,
accounted for. A representative HPLC radiochromatogram of rat plasma it was important to determine whether the exposure of these two metab-
is shown in Fig. 7B. Of the four radioactive peaks identified in the olites was greater than or similar to the exposure in the toxicology

METABOLISM AND EXCRETION OF ZONIPORIDE
647
TABLE 3
Percentage of urinary and fecal metabolites of zoniporide after intravenous administration of [14C]zoniporide to healthy male volunteers, Sprague-Dawley rats, and
Beagle dogs
Healthy male volunteers were administered ͓14C͔zoniporide by intravenous infusion for 1 h at a dose of 80 mg (100 ␮Ci). The rats were administered a bolus of [14C]zoniporide at a dose
of 10 mg/kg (50 ␮Ci). The dogs were administered a bolus of ͓14C͔zoniporide at a dose of 3 mg/kg (150 ␮Ci).
% Dose Excreted
Metabolites
Healthy Male Volunteers
Feces
Sprague-Dawley Rats
Beagle Dog
Feces
Urine
16
Total
Urine
10
Feces
25
Total
35
Urine
Total
M1
M2
36
17
52
17
M3
M4
M5
M6
M7
M8
M9
M10
Zoniporide
0.8
2.1
0.76
1.4
0.8
2.1
2.1
1.4
5.9
0.7
1.2
7.9
4.1
8.6
5.4
1.3
5.9
4.1
11
4.1
13
2.1
18
1.7
2.1
0.29
18
18
19
17
1.0
18
12
28
40
37
A
zoniporide using this method resulted in AUC0–tlast values of 878,
1285, and 1959 ng-Eq-h/ml in humans, rat, and dog, respectively,
which were within the range of experimental error compared with the
levels that were determined by a validated bioanalytical method
(Table 2).
To further confirm the coverage of M1 and M3 in the preclinical
species, the exposures of these two metabolites were also estimated
using eq. 5, and the results are depicted in Table 5:
1600
1400
1200
1000
800
M1
Zoniporide
M6
M5
600
M4
400
M3
200
Estimated exposuremetabolite
0
ϭ (% radioactivitymetabolite/% radioactivityparent) ϫ AUCzoniporide (5)
0.0
10.0
20.0
Time (min)
30.0
40.0
50.0
B
3000
2500
2000
1500
1000
500
Zoniporide
A
Zoniporide
3000
2500
2000
1500
1000
500
M9
M1
M8
M7
M5
M3
0
0.0
10.0
20.0
30.0
40.0
50.0
0
Time (min)
0.0
10.0
20.0
30.0
40.0
50.0
FIG. 5. Representative HPLC radiochromatogram of pooled urine (A) and feces (B)
after intravenous administration of [14C]zoniporide at a dose of 10 mg/kg (50 ␮Ci)
to Sprague-Dawley rats.
Time (min)
B
3000
2500
2000
1500
1000
500
M8
species. To assess the coverage of M1 and M3, the exposures of these two
metabolites in humans and preclinical species were estimated by eq. 4:
M10
Zoniporide
M3
AUC(0 –tlast)metabolite ϭ % of metabolite ϫ AUC(0 –tlast)radioactivity (4)
M7
The estimated exposures of all metabolites are depicted in Table 4. As
shown in Table 4, the exposures (AUC0–tlast) of M1 and M3 were
1755 and 187 ng-Eq-h/ml, respectively, in humans and the estimated
exposures of M1 and M3 in the rat were 1642 and 321 ng-Eq-h/ml,
respectively. Metabolite M1 was not present in the dog; however, the
estimated AUC(0–tlast) of M3 in this species was 73 ng-Eq-h/ml. The
estimated exposures of other metabolites observed in the rat and dog
0
0.0
10.0
20.0
Time (min)
30.0
40.0
50.0
FIG. 6. Representative HPLC radiochromatogram of urine (A) and feces (B) after
intravenous administration of [14C]zoniporide at a dose of 3 mg/kg (150 ␮Ci) to
are also shown in Table 4. Determination of circulating levels of Beagle dogs.

648
DALVIE ET AL.
amount of metabolite excreted in the urine and feces of humans and
A
M1
600
rats, which provides a comparison of total body burden. The amount
of M1 excreted in the urine and feces was determined using eq. 6:
500
400
Amount of MI (␮g/kg) ϭ dose (mg/kg) ϫ % MI in the excreta (6)
Zoniporide
300
200
As shown in Table 6, the amount of M1 in the excreta of humans
after 1.14 mg/kg (assuming a 70-kg weight for humans) and rats after
a 10 mg/kg dose was 594 and 3500 ␮g/kg, respectively. Thus, even
though the circulating exposure to M1 in the rat is slightly lower than
that in humans, the total body exposure in the rat is actually greater.
Identification of Metabolites. The structures of metabolites were
elucidated by LC-MS/MS using a combination of full, precursor ion,
and neutral loss scanning techniques. All metabolites were further
characterized using the product ion scans of the identified masses.
Zoniporide gave a signal at m/z 321 [M ϩ H]ϩ in a positive ion mode.
The product ion mass spectrum of m/z 321 gave characteristic major
fragment ions at m/z 262 (loss of guanidine) and 234 (further loss of
carbon monoxide) and 220 (loss of the cyclopropyl ring) (Table 7).
Wherever possible, the structures of the major metabolites were
confirmed by comparing their retention time and mass spectra with
those of the synthetic standards. The structures/proposed structures of
metabolites of zoniporide are shown in Fig. 8.
Metabolite M1. Metabolite M1 showed a molecular ion at m/z 337,
which was 16 amu higher than zoniporide suggesting oxidation of the
parent compound. The mass spectrum of m/z 337 gave a fragment ion
at m/z 278, 250, and 236 (Table 7). All of these ions were 16 amu
greater than those observed in the mass spectrum of zoniporide. This
finding suggested that the quinoline or the pyrazole moiety of the
molecule was the site of metabolism. That the site of hydroxylation
was the quinoline ring and M1 was 2-oxozoniporide was confirmed
by comparing its retention time and mass spectrum with the authentic
standard of 2-oxozoniporide.
M3
100
0
0
10
20
30
40
50
Time (min)
M1
B
3000
2500
2000
1500
1000
500
Zoniporide
M3
M6
0
0.0
10.0
M9
20.0
30.0
40.0
50.0
Time (min)
C
Zoniporide
1400
1200
1000
800
600
M3
400
M8
M4
M10
200
0
0
10
20
30
Time (min)
40
50
Metabolite M2. Metabolite M2 gave a molecular ion at m/z 296.
The mass spectrum of M2 at m/z 296 resulted in fragment ions at m/z
278 and 250 (Table 7). The ion at m/z 278 was a result of loss of water
from M2, and the fragment ion at m/z 250 was a result of a loss of 46
amu from m/z 296. The loss of 46 amu, which is characteristic of a
FIG. 7. HPLC radiochromatogram of pooled plasma of healthy male volunteers
after intravenous infusion of 80 mg [14C]zoniporide for 1 h (A) and of Sprague-
Dawley rats (B) and Beagle dogs (C) after intravenous administration of [14C]zo-
niporide at a dose of 10 and 3 mg/kg, respectively.
Because both human circulating metabolites, M1 and M3, were carboxylic acid group from the molecule, suggested that M2 was a
present in disproportionately lesser amounts in the dog, an assessment carboxylic acid derivative. The presence of the carboxylic acid was
of the coverage of exposure of these two metabolites was made only further confirmed by esterification with methanolic HCl to the
in the rat. Using eq. 2, the exposure of M1 and M3 was 1666 and 178 corresponding methyl ester (data not shown). The mass spectral
ng-Eq-h/ml, respectively, in humans and 1776 and 348 ng-Eq-h/ml in fragmentation also indicated that the molecular ion of M2 was 16
the rat.
amu higher than that of M3 (see below). Overall, the above data
Assessment of coverage of M1 was also made by comparing the indicated that M2 was a carboxylic acid analog of the hydroxylated
TABLE 4
Percentage of circulating metabolites of zoniporide and their estimated exposure in healthy male volunteers, Sprague-Dawley rats, and Beagle dogs after intravenous
administration of [14C]zoniporide
Healthy male volunteers were administered ͓14C͔zoniporide by intravenous infusion for 1 h at a dose of 80 mg (100 ␮Ci). The rats were administered a bolus of ͓14C͔zoniporide at a dose
of 10 mg/kg (50 ␮Ci). The dogs were administered a bolus of ͓14C͔zoniporide at a dose of 3 mg/kg (150 ␮Ci). The estimated exposures were determined by multiplying percent circulating
radioactivitymetabolite and AUC(0–tlast)radioactivity. The AUC(0–tlast)radioactivity is depicted in Table 2.
Human
Circulating Radioactivity
Rat
Dog
Circulating Radioactivity
Metabolites
Estimated AUC(0–tlast)
Circulating Radioactivity
Estimated AUC(0–tlast)
Estimated AUC(0–tlast)
%
ng-Eq-h/ml
%
ng-Eq-h/ml
%
ng-Eq-h/ml
M1
M3
60
6.4
1755
187
46
9.0
1642
321
3.7
2.3
117
73
M4
M6
M8
M9
M10
Zoniporide
8.5
36
303
5.4
171
727
98
23
3.1
62
30
878
1285
1959

METABOLISM AND EXCRETION OF ZONIPORIDE
649
TABLE 5
Metabolite M5. Metabolite M5 showed a protonated molecular ion at
m/z 353, which was 32 amu greater than the molecular ion of zoniporide
(m/z 321). Addition of 32 amu to the parent suggested further dihydroxy-
lation of zoniporide. The mass spectrum of the metabolite showed major
fragment ions at m/z 336, 294, 277, 266, and 249 (Table 7). The fragment
ion at m/z 294 and 266 indicated a loss of 59 amu (loss of the guanidine
moiety) followed by the loss of a carbonyl group, suggesting that the
pyrazoloquinoline moiety of zoniporide was dihydroxylated. Other frag-
ment ions m/z 277 and 249, observed in the CID spectrum, indicated a
loss of 17 amu (hydroxyl group) from m/z 294 and 266, respectively.
Because the loss of 17 amu is characteristic of an N-oxide as discussed
above, M5 was proposed as an N-oxide of hydroxyzoniporide. No further
characterization of this metabolite was done to ascertain the position of
the hydroxyl group because the metabolite was present only in the
preclinical species.
Estimation of exposure of metabolites M1 and M3 in rat plasma
Metabolite/parent ratio was determined from the abundance of the metabolite (% total
circulating radioactivity) and parent (% total circulating radioactivity) in the plasma.
Estimated exposure of metabolite ϭ (metabolite/parent ratio) ⅐ AUCzoniporide. The AUC of
zoniporide was determined after quantitation of the parent by a bioanalytical assay.
Human
Rat
Metabolite
M/P Ratio
Estimated Exposure
M/P Ratio
Estimated Exposure
M1
M3
2
0.21
1666
178
1.3
0.25
1776
348
M/P, metabolite/parent.
TABLE 6
Estimation of total body burden of M1 from excretion data in humans and rats
Amount of M1 in the excreta was determined using eq. 3.
Metabolite M6. Metabolite M6 showed a protonated molecular ion
at m/z 513, which was 176 amu higher than m/z 337, the hydroxylated
zoniporide. This suggested glucuronidation of hydroxyzoniporide.
The mass spectrum of m/z 513 gave a major fragment ion at m/z 337,
suggesting a loss of 176 amu from the molecular ion, and a fragment
ion at m/z 278, suggesting further loss of guanidine. Based on these
data, M6 was identified as the glucuronide conjugate of hydroxyzo-
niporide. The position of the hydroxy group could not be assessed
Estimation of Metabolite M1
Dose
Dose
M1
Amount
mg
mg/kg
% dose
␮g/kg
Humans
Rats
80
N.A.
1.14
10
52
35
594
3500
N.A., not available.
metabolite of zoniporide. The position of the hydroxyl group on from these data. No attempt was made to further characterize this
the molecule and its structure was further confirmed by comparing metabolite because it was absent in the human matrices.
its retention time and mass spectrum with the synthetic standard
of M2.
Metabolite M7. Metabolite M7 showed a protonated molecular ion
at m/z 337, suggesting hydroxylation of zoniporide. The fragment ions
Metabolite M3. Metabolite M3 gave a signal at m/z 280 (41 amu in the mass spectrum of the metabolite were similar to those observed
less than the parent). A mass spectrum of M3 at m/z 280 showed major in the mass spectrum of metabolite M1 (Table 7). The difference in
fragment ions at m/z 262 and 234, suggesting a loss of a water the retention time of M1 suggested that M7 was a regio-isomer of M1;
molecule followed by a loss of carbonyl functionality (Table 7). A however, the exact position of the hydroxyl group could not be
difference of 46 amu between m/z 280 and m/z 234 suggested a loss determined from the mass spectrum. Because the oxidative metabolite
of formic acid, which was characteristic of a carboxyl group. Esteri- was lacking in humans, no further attempt was made to determine the
fication of the metabolite using methanolic HCl resulted in the for- position of hydroxylation in the metabolite.
mation of methyl ester, further confirming the presence of the car-
Metabolite M8. Metabolite M8 showed a molecular ion at m/z 355.
boxylic acid group in the molecule. Furthermore, these ions were The molecular ion of M8 indicated an addition of 34 amu to m/z 321, a
similar to those observed in the mass spectrum of zoniporide, sug- molecular ion of zoniporide. The mass spectrum of m/z 355 gave a
gesting that the guanidine moiety of zoniporide was modified (Table fragment ion at m/z 296 (a characteristic loss of the guanidine moiety) and
7). Together the above data suggested that M3 was a carboxylic acid at m/z 296, 278, 254, and 250 (Table 7). The loss of 59 amu that resulted
derivative of zoniporide. Comparison of the retention time and mass in m/z 296 indicated that the guanidine moiety was intact. The fragment
spectrum of M3 with that of the authentic standard of carboxylic acid ion at m/z 278 and 250 possibly resulted from loss of water from
further confirmed its structure.
m/z 296 followed by a loss of the carbonyl group (28 amu). The
Metabolite M4. Metabolite M4 showed a molecular ion at m/z 337, mass spectrum also showed ions at m/z 254 and 236, suggesting a
which was 16 amu greater than zoniporide. The mass spectrum of m/z loss of 42 amu (a cyclopropyl group) from m/z 296 and 278,
337 resulted in fragment ions at m/z 320, 278, 261, and 250 (Table 7). respectively. The loss was similar to that observed in the CID
The fragment ions at m/z 278 and 250 were similar to those observed spectrum of zoniporide and suggested that the pyrazoloquinoline
in the mass spectrum of M1 and resulted from the loss of guanidine group was the site of modification. Together, these fragment ions
(59 amu) followed by the loss of the carbonyl moiety (28 amu), indicated that the M8 was probably a dihydrodiol metabolite of
suggesting that the addition had occurred on the pyrazolo-quinoline zoniporide (Fig. 8). Previous studies with quinoline-containing
moiety. The fragment ions at m/z 320 and 261 resulted from the loss compounds have indicated that these motifs are capable of under-
of 17 amu (a hydroxyl group) from m/z 337 and 278, respectively, and going conversion to a dihydrodiol (Nicoll-Griffith et al., 1993).
characteristic of an N-oxide. Treatment of the samples with 33% The definitive identification and characterization of the exact po-
titanium chloride (TiCl3) (Kulanthaivel et al., 2004) resulted in the sition of the dihydrodiol on the quinoline ring was not attempted.
disappearance of the peak, which suggested that the metabolite was an
Metabolite M9. Metabolite M9 showed a molecular ion at m/z 531,
N-oxide of zoniporide. The exact location of the oxygen atom (the which was 176 amu higher than M8, indicating glucuronidation of
quinoline nitrogen or pyrazole nitrogen) could not be discerned from M8. The mass spectrum showed fragment ions at m/z 472, 355, 296,
the data. However, because previous studies have shown that quino- 278, and 250 (Table 7). The fragment ion at m/z 472 was a charac-
line-containing molecules can also undergo oxidation of the quinoline teristic loss of the guanidine moiety, suggesting that this group was
ring nitrogen in addition to the ring (Ehlhardt et al., 1998), the not modified. The fragment ion at m/z 355 resulted from the loss of the
structure of M4 was proposed as the quinoline N-oxide of zoniporide. glucuronide moiety. The remaining fragment ions were similar to
No further studies were attempted to isolate and fully characterize the those in the mass spectrum of M8. Previous studies have demonstrated
metabolite.
that dihydrodiol metabolites of aromatic rings are capable of under-

650
DALVIE ET AL.
TABLE 7
Mass spectral fragmentation and structures of zoniporide and proposed metabolites
ϩ
Structure
͓M ϩ H͔
Fragment Ions
HN
Zoniporide
321
262
278
234
220
262
O
- CO
NH2
NH
234
N
N
220
HN
N
M1
337
250
236
278
O
- CO
NH2
NH
250
N
N
236
O
O
N
H
M2
296
278
250
278
250
OH
N
N
O
N
H
M3
M4
280
337
262
320
234
278
262
234
O
OH
N
N
N
261
250
-OH
HN
278
261
O
NH2
-CO
NH
250
N
N
320
N
O
M5
353
336
294
277
266
-OH
HN
294
277
O
NH2
-CO
NH
266
N
N
336
OH
N
O
M6
513
337
278
HN
278
O
NH2
337
NH
N
N
O
O
CO2H
OH
N
OH
HO
]
M7 and M10
337
278
250
HN
278
- CO
O
NH2
NH
250
N
N
OH
N
Continued

METABOLISM AND EXCRETION OF ZONIPORIDE
651
TABLE 7—Continued
ϩ
Structure
͓M ϩ H͔
Fragment Ions
254
M8
355
296
278
250
236
HN
- H2O 296
278
O
NH2
254
NH
250
N
N
236
N
OH
OH
296
M9
531
472
355
296
278
HN
NH
O
- H2O
NH2
278
N
N
Glucuronide
N
OH
OH
355
going glucuronidation (Lantz et al., 2003). Thus, M9 was assumed to those observed in the mass spectrum of metabolite M1 and M7 (Table
be a glucuronide conjugate of M8 (Fig. 8). No further characterization 7). The difference in the retention time of M10 from that of M1 and
of this metabolite was performed because it was not detected in the M7 suggested that M10 was an isomer of these two metabolites;
human metabolic profiles.
however, the exact position of the hydroxyl group could not be
Metabolite M10. Metabolite M10 also showed a protonated molec- determined from the mass spectrum. Because an oxidative metabolite
ular ion at m/z 337, suggesting hydroxylation of zoniporide. The was lacking in humans, no further attempt was made to determine the
fragment ions in the mass spectrum of the metabolite were similar to position of hydroxylation in the metabolite.
O
OH
N
N
M2
O
N
H
HN
O
HN
NH2
O
O
NH
NH2
OH
NH
N
N
N
N
N
N
O
N
H
N
N
M3
M1
Zoniporide
HN
HN
FIG. 8. Proposed metabolic scheme of zoniporide in healthy male
volunteers, Sprague-Dawley rats, and Beagle dogs after intravenous
administration.
O
NH2
O
HN
NH2
NH
O
NH
NH2
NH
N
N
N
N
N
N
N
OH
N
O
OH
OH
M4
N
M8
M7
HN
HN
HN
O
O
NH2
NH2
O
NH2
NH
NH
NH
N
N
N
N
N
N
Glucuronide
OH
M5
O
CO2H
N
O
N
OH
N
OH
O
OH
OH
M9
HO
M6

652
DALVIE ET AL.
M1
A
337.11
A
100
Zoniporide
80
60
40
20
WO NADPH
0.06
321.18
0.05
0.04
0.03
0.02
0.01
0.00
337.12
B
100
80
M1
W NADPH
Zoniporide
321.12
60
40
20
321.11
C
100
80
Zoniporide
W Raloxifene
60
0
100
200
300
400
500
40
Zoniporide (µM)
M1
20
B
120
100
80
60
40
20
0
17
18
19
20
21
Time (min)
22
23
24
25
FIG. 9. Metabolic profile of zoniporide (10 ␮M) after incubation with human liver
S9 fractions in the presence (W) of NADPH (A), absence (WO) of NADPH (B), and
presence (W) of raloxifene, an inhibitor of aldehyde oxidase (C).
In Vitro Metabolism Studies. To identify the enzymes responsible
for the formation of M1, preliminary experiments were performed by
incubating zoniporide with human liver S9 fraction at concentrations
of 10 ␮M in the presence and absence of NADPH (Fig. 9, A and B).
The presence of M1 in the incubations that lacked NADPH suggested
that its formation was catalyzed by a non-P450 enzyme. Furthermore,
inhibition of M1 in the presence of raloxifene, an aldehyde oxidase
inhibitor, to the incubation mixture, indicated that aldehyde oxidase
was responsible for its formation (Fig. 9C). Only trace amounts of M3
were detected in the mass spectral analysis of the above incubation
mixture (data not shown) suggesting that hydrolysis constituted a
minor pathway at least in incubations with liver S9 fractions.
Based on the above information, enzyme kinetics studies for for-
mation of M1 by aldehyde oxidase were performed using pooled
human liver cytosol, and the kinetic parameters were determined.
Analysis of the relationship between reaction velocity and substrate
concentration revealed substrate inhibition kinetics with kinetic pa-
rameters of Km ϭ 3.4 Ϯ 0.2 ␮M and Vmax ϭ 74 Ϯ 2 pmol/min/mg
protein and a Ks value of 152 ␮M (Fig. 10A). A detailed assessment
of the molybdenum hydrolases (aldehyde oxidase or xanthine oxi-
dase) was also conducted by determining the IC50 for the inhibition of
M1 formation using specific inhibitors of aldehyde oxidase (ralox-
ifene and menadione) and xanthine oxidase (allopurinol). Raloxifene
potently inhibited the reaction (IC50 ϭ 0.012 Ϯ 0.002) and menadione
also inhibited the reaction (9.8 Ϯ 1.2 ␮M), further confirming the
involvement of aldehyde oxidase in M1 formation (Fig. 10B). Allo-
purinol did not inhibit the reaction (IC50 Ͼ 1 mM), indicating that
xanthine oxidase did not metabolize zoniporide.
raloxifene
menadione
allopurinol
0.001
0.01
0.1
1
10
100
1000
Inhibitor (µM)
FIG. 10. Enzyme kinetics for the formation of 2-oxozoniporide (M1) from zonipo-
ride in pooled human liver cytosol (A) and inhibition of this reaction by raloxifene,
menadione, and allopurinol (B). The enzyme kinetics experiments were done in
triplicate, and the inhibition studies were done in duplicate. The errors bars in A
indicate S.D.
comparison of metabolite profiles of a drug candidate in animals and
humans is essential to ensure that animal species used in toxicological
evaluations are appropriate models of humans and to confirm that all
human circulating metabolites are covered in these species (Food and
Drug Administration, 2008). The metabolic profiles in humans, rat,
and dog were compared to assess whether all metabolites observed in
human matrices were detected in these two toxicology species. The
doses used in the preclinical species were equivalent to the dose in the
toxicology studies in which minimum adverse effects were observed.
After intravenous administration, the majority of the radioactivity
was excreted in feces, suggesting that biliary excretion was the prin-
cipal route of elimination of zoniporide-related material in the hu-
mans. Metabolism was the primary route of clearance for zoniporide
in humans because only 18% of the dose was excreted unchanged in
the urine and feces. In addition, the majority of the total circulating
radioactivity comprised metabolites because the exposure of un-
Discussion
In the present study, the routes of elimination and metabolism and
the excretion mass balance of zoniporide in humans were investigated
after an intravenous infusion of [14C]zoniporide. A dose of 80 mg was
administered in this study, which was approximately equal to the changed zoniporide accounted for ϳ28% of exposure of total circu-
anticipated clinical dose to be used in the phase III program. The lating radioactivity in plasma. 2-Oxozoniporide (M1), which was

METABOLISM AND EXCRETION OF ZONIPORIDE
653
formed via oxidation of the quinoline ring of zoniporide, was the humans, zoniporide metabolism was the primary route of clearance in
primary circulating and excretory metabolite in humans. Its exposure
was 2-fold greater than that of unchanged zoniporide in plasma, and
approximately 52% of the dose was excreted as M1 in the urine and
feces. Metabolite M1 was also pharmacologically active against the
NHE-1 receptor. However, the activity was ϳ3-fold less than that of
the parent compound. Because the systemic exposure of M1 was
greater than that of the parent drug and the protein binding was
approximately the same as that of zoniporide, the possibility of the
metabolite exerting pharmacological activity in addition to the parent
in vivo could not be ruled out. Given that M1 was the major circu-
lating metabolite in humans and was pharmacologically active, in
vitro studies using human liver subcellular fractions were performed
to identify the enzymes responsible for the formation of M1. Further-
more, this was important in light of the fact that inhibition of the
enzyme responsible for the conversion of zoniporide to M1 would
increase the AUC of the parent drug significantly and would also
possibly affect the efficacy of the compound. Phenotying studies
revealed that formation of M1 was primarily catalyzed by aldehyde
oxidase (Clint ϭ 22 ␮l/min/mg cytosolic protein). Aldehyde oxidase is
a cytosolic molybdo-flavoenzyme that is expressed predominantly in
the liver, lung, and kidney and plays a major role in the oxidation of
aldehydes and nitrogen-containing heterocyclic compounds (Kita-
mura et al., 2006). Some heteroaromatic compounds of pharmacolog-
ical and toxicological importance that are metabolized by aldehyde
oxidase include carbazeran (Kaye et al., 1985), famciclovir (Rashidi et
al., 1997), methotrexate (Jordan et al., 1999), zaleplon (Lake et al.,
2002), brimonidine (Acheampong et al., 1996), and N-[(2Ј-diethyl-
amino)ethyl]acridine-4-carboximide (Schofield et al., 2000). Thus, it
is not surprising that zoniporide was a substrate of aldehyde oxidase.
the rat and dog. Only 40 and 37% of the total dose constituted
unchanged zoniporide in the rat and dog, respectively. However,
comparison of plasma exposure of unchanged zoniporide and total
radioactivity in rat and dog revealed that even though circulating
radioactivity comprising metabolites in rats just as in humans, un-
changed zoniporide was the primary constituent of total radioactivity
in dogs.
Analysis of the metabolic profile of zoniporide in the rat revealed
that both human circulating metabolites (M1 and M3) were present in
the rat in addition to several other metabolites. Although dogs me-
tabolized zoniporide extensively, in contrast to humans and rats, this
species showed differences in its metabolic profile. Metabolite M1,
which was a major metabolite in rats and humans, was not detected in
the dogs, whereas M3 was a minor circulating metabolite (6% of the
parent) but present in ϳ9% of the dose in the excreta. The absence of
M1 in the dog was not surprising given that dogs lack aldehyde
oxidase activity (Kitamura et al., 2006). Even though oxidation was a
primary pathway of metabolism in dogs, the major metabolites ob-
served were the dihydrodiol (M8) and hydroxyzoniporide (M10).
Because the other oxidative metabolites detected in the rat and dog
were absent in humans, no further consideration was given to these
metabolites with respect to potential safety implications.
An assessment of the coverage of major circulating metabolites in
humans was made in this study by comparing estimated exposure
levels of M1 and M3 in humans with those in preclinical species.
Assessment was done only in the rat because the dog did not produce
the M1 metabolite, and M3 was produced in Ͻ10% of the parent in
vivo. Because levels of M3 in rat exceeded those estimated in humans
Potential drug-drug interactions due to inhibition of aldehyde oxidase by 1.7- to 2.0-fold (Table 4), the exposure to this human metabolite
have not been established. Although cytochrome P450 enzymes have
been and continue to be a major focus of drug interactions, alterations in
the activities of other drug-metabolizing enzymes can also be an under-
lying mechanism of drug-drug interactions. Only one clinically relevant
drug-drug interaction between cimetidine and zaleplon has been ascribed
to inhibition of aldehyde oxidase in the literature so far (Renwick et al.,
2002). Because several drugs have been demonstrated to be human
aldehyde oxidase inhibitors in vitro (Obach, 2004; Obach et al., 2004), it
is possible that these drugs could potentially increase the levels of
zoniporide in humans. No such drug-drug interactions studies have been
conducted and it remains to be determined whether aldehyde oxidase
inhibitors identified using in vitro methods could potentially cause a more
profound interaction of clinical significance in vivo.
Metabolites M3 and M2, the carboxylic acid analogs of zoniporide
and quinolone metabolite (M1), were also observed in humans. This
finding suggested that the hydrolytic cleavage was another pathway
by which zoniporide was metabolized. Although M3 accounted for
ϳ20% of parent drug in humans, M2 was found only in the excreta in
amounts of ϳ17% of the dose. The exact metabolic pathway for the
formation of M2 was difficult to discern because the metabolite can be
formed via two parallel pathways (Fig. 8). One pathway could involve
hydrolysis of M1 to M2. Alternatively, zoniporide could undergo
hydrolysis to M3, which could subsequently undergo an aldehyde
oxidase-mediated or a P450-mediated oxidation of the quinoline ring
to yield M2. Because M2 was not detected in the urine or the plasma
of humans, it is possible that the formation of this metabolite in vivo
was possibly mediated by microflora in the gastrointestinal tract.
The metabolism and excretion studies using [14C]zoniporide in the
rat and dog suggested that the route of excretion of zoniporide in these
two species was similar to that in humans and the majority of the dose
was considered to be covered in the rat. The estimated exposure level
of M1 in humans, on the other hand, was comparable with that
estimated in rats as depicted in Tables 4 and 5. Because zoniporide
was administered intravenously to humans and the preclinical species,
the coverage of metabolite M1 in the preclinical species was also
ascertained by estimating the amount of metabolite excreted in the rat
and human. This estimation indicated that the amount of M1 to which
the rat was exposed was 5.9-fold greater relative to that in humans.
Furthermore, M1 did not have any structural alerts that would pose a
risk to humans. Thus, M1 was also considered to be adequately evalu-
ated in the toxicology species, and no further assay validation and
toxicological evaluation of this metabolite was undertaken in nonclinical
safety species. Metabolite M2 was detected only in human feces and was
not present in rat and dog, which indicated that this metabolite was
unique to the humans. However, because M2 was not present in circu-
lation, was pharmacologically inactive, and did not have a functionality
that could potentially be a toxicophore, it was not considered for further
safety evaluation.
In summary, this study demonstrates the primary routes of excre-
tion and metabolism of zoniporide in humans, rats, and dogs. Fur-
thermore, the study also demonstrates the strategy that was used to
assess whether the further toxicological evaluation of the human
circulating metabolites was necessary in the preclinical species. Zo-
niporide is among the few compounds that are almost exclusively
metabolized by aldehyde oxidase in humans (with approximately
three-fourths of its clearance mediated by this enzyme) and hence can
be used as a model substrate for this enzyme. Studies are in progress
to assess the species differences in the metabolism of aldehyde oxi-
dase and understand the structure-metabolism relationship of the
was also eliminated into the feces via the bile. In addition, as in compound.

654
DALVIE ET AL.
inhibitor, zoniporide, reduces ischemic myocardial injury in vitro and in vivo. J Pharmacol
Exp Ther 297:254–259.
Acknowledgments. We thank the radiochemistry group in Groton
for synthesizing [14C]zoniporide.
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