METABOLISM OF VABICASERIN IN MICE, RATS,...

1

METABOLISM OF VABICASERIN IN MICE, RATS, DOGS, MONKEYS AND HUMANS

Zeen Tong, Appavu Chandrasekaran, William DeMaio, Robert Espina, Wei Lu, Ronald Jordan,

and JoAnn Scatina

Pfizer Inc., 500 Arcola Road, Collegeville, PA 19426.

DMD # 33670 DMD Fast Forward. Published on August 25, 2010 as doi:10.1124/dmd.110.033670

Copyright 2010 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on August 25, 2010 as DOI: 10.1124/dmd.110.033670

at ASPE

T Journals on A

ugust 4, 2018dm

d.aspetjournals.orgD

ownloaded from

http://dmd.aspetjournals.org/

2

Running title: Metabolism of vabicaserin

Corresponding author:

William DeMaio

4 Wynnewood Dr

Collegeville, PA 19426

Phone: (484)902-0306

Fax: none

E-mail: [email protected]

Numbers of pages:

Number of tables: 5

Number of Figures: 10

Number of references: 19

Number of words: Abstract: 237; Introduction: 402; Discussion: 700.

DMD # 33670This article has not been copyedited and formatted. The final version may differ from this version.

DMD Fast Forward. Published on August 25, 2010 as DOI: 10.1124/dmd.110.033670 at A

SPET

Journals on August 4, 2018

dmd.aspetjournals.org

Dow

nloaded from


3

Abstract

Vabicaserin is a potent 5-HT2C agonist that is currently being developed for the treatment of the

psychotic symptoms of schizophrenia. In this study, in vitro and in vivo metabolism of

vabicaserin was evaluated in mice, rats, dogs, monkeys and humans, and the structures of the

metabolites were characterized by LC/MS and NMR spectroscopy. Vabicaserin underwent three

major metabolic pathways in vitro: NADPH-dependent hydroxylation, NADPH-independent

imine formation and carbamoyl glucuronidation. Following a single oral dose, vabicaserin was

extensively metabolized in animals and humans, and its metabolites were mainly excreted via the

urine in mice and rats. Along with the metabolites observed in vitro, secondary metabolism via

oxidation and conjugation of the primary metabolites generated from the above mentioned three

pathways yielded a number of additional metabolites in vivo. Carbamoyl glucuronidation was

the major metabolic pathway in humans, but a minor pathway in rats. Although carbamoyl

glucuronidation was a major metabolic pathway in mice, dogs and monkeys, oxidative

metabolism was also extensive in these species. Hydroxylation occurred in all species, although

different regional selectivity was apparent. The imine pathway also appeared to be common to

several species, since vabicaserin imine was observed in humans and hydroxyl imine metabolites

were observed in mice, rats and dogs. A nitrone metabolite of vabicaserin was observed in dogs

and humans but not in other species. In conclusion, the major metabolic pathways for

vabicaserin in humans and non-clinical safety species include carbamoyl glucuronidation,

hydroxylation, formation of an imine and a nitrone.



SPET



Dow

nloaded from


4

Introduction

Recent studies have demonstrated that 5-HT2C agonists decrease levels of dopamine in the

prefrontal cortex and nucleus accumbens, brain regions that are thought to mediate the critical

effects of antipsychotic drugs (Millan et al., 1998; DiMatteo et al., 1999; DiGiovanni et al.,

2000). In contrast, 5-HT2C agonists do not decrease dopamine levels in the striatum, the brain

region most closely associated with extrapyramidal side effects (Millan et al., 1998; DiMatteo et

al., 1999). In addition, a recent study demonstrated that 5-HT2C agonists decrease firing in the

ventral tegmental area but not in the substantia nigra (DiMatteo et al., 1999; DiGiovanni et al.,

2000). The differential effects of 5-HT2C agonists in the mesolimbic pathway relative to the

nigrostriatal pathway suggests that 5-HT2C agonists have the potential to treat psychotic

symptoms with lower liability for the extrapyramidal side effects associated with typical

antipsychotics.

Vabicaserin is a potent 5-HT2C full agonist and demonstrates in vitro functional selectivity for 5-

HT2C over 5-HT2A and 5-HT2B receptors (Dunlop et al., 2006). Vabicaserin is effective in

several animal models that are predictive of antipsychotic activity, with an atypical antipsychotic

profile (Marquis et al., 2006). Administration of vabicaserin decreases nucleus accumbens

dopamine levels without affecting striatal dopamine, which is indicative of mesolimbic

selectivity. This profile is consistent with potential efficacy in the treatment of the psychotic

symptoms of schizophrenia with decreased liability for extrapyramidal side effects. In addition,

chronic administration of vabicaserin significantly decreases the number of spontaneously active

mesocorticolimbic dopamine neurons, without affecting nigrostriatal dopamine neurons,

consistent with the effects of atypical antipsychotics.



SPET



Dow

nloaded from


5

Formation of a carbamoyl glucuronide (CG) was the major metabolic pathway in human liver

microsomes with bicarbonate buffer and a CO2-enriched environment in the presence of both

NADPH and UDPGA (Tong et al., 2010). Following a single oral dose to healthy human

volunteers, the carbamoyl glucuronide (CG) was the predominant metabolite in human plasma

and urine, with average CG-to-vabicaserin concentration ratios ranging from 8 to 74 in plasma

and 96 to 537 in urine (Tong et al., 2010). However, oxidative metabolism also appeared to be

occurring in vitro in liver microsomes (Tong et al., 2010). Although, the in vivo formation of

oxidative metabolites was evident from their presence in plasma and urine, the structures of

those metabolites were previously not established due to limited availability of human biological

samples (Tong et al., 2010). Therefore, the purpose of the current study was to generate and

isolate sufficient amounts of these oxidative metabolites, elucidate the structures of the major

vabicaserin metabolites by LC/MS and NMR spectroscopy, determine mass balance in the safety

species and compare in vitro and in vivo metabolite profiles of vabicaserin in humans and non-

clinical safety species.



SPET



Dow

nloaded from


6

Materials and Methods

Materials. [14C]Vabicaserin hydrochloride {(9aR, 12aS)-4, 5, 6, 7, 9, 9a, 10, 11, 12, 12a-

decahydro-cyclopenta[c][1, 4]diazepino[6, 7, 1-ij]quinoline} was synthesized by the

Radiosynthesis Group, Chemical Development, Wyeth (now Pfizer, Pearl River, NY). The

radiochemical purity of [14C]vabicaserin was 98.9% and the chemical purity was 99.9% by UV

detection. The specific activity of the [14C]vabicaserin was 222.9 μCi/mg as a hydrochloride

salt. Non-labeled vabicaserin hydrochloride with a chemical purity of 98.6% was synthesized by

Wyeth (Pearl River, NY). Vabicaserin CG (WAY-280107) was synthesized by Chemical

Development at Wyeth (Montreal, Quebec), and had a purity of 95.5%. The chemical structures

of [14C]vabicaserin and its CG are shown in Figure 1. Liver microsomes listed in Table 1 from

CD-1 mice, Sprague-Dawley rats, beagle dogs and cynomolgus monkeys were obtained from In

Vitro Technologies (Baltimore, MD). Pooled human liver microsomes from 10 subjects of

mixed sex were purchased from Xenotech, LLC (Lenexa, KS) (Table 1). Glucose-6-phosphate,

glucose-6-phosphate dehydrogenase, nicotinamide adenine dinucleotide phosphate (NADP+),

uridine 5′-diphosphoglucuronic acid trisodium salt (UDPGA), EDTA, 3-chloroperoxybenzoic

acid (mCPBA, 77%), silica gel (230-400 mesh) and Fluka silica gel TLC plates were obtained

from Sigma-Aldrich (St. Louis, MO). Acetonitrile, ethyl acetate and methanol were HPLC or

ACS reagent grade and were purchased from EMD Chemicals (Gibbstown, NJ). Ultima Gold

and Ultima Flo scintillation cocktails were purchased from PerkinElmer (Wellesley, MA).

DMSO-d6 (D, 99.96%) was obtained from Cambridge Isotope Laboratories (Andover, MA).

Other chemicals of analytical grade and solvents of high performance liquid chromatography

(HPLC) grade were obtained from EMD Chemicals (Gibbstown, NJ) or Mallinckrodt Baker Inc.

(Phillipsburg, NJ).



SPET



Dow

nloaded from


7

Vabicaserin nitrone was synthesized for NMR spectroscopic analysis. At room temperature with

stirring, a solution of mCPBA (216 mg, about 1 mmol) in acetonitrile (75 mL) was slowly added

to a suspension of vabicaserin (230 mg, 1.0 mmol) in acetonitrile (100 mL) and kept stirring at

room temperature for 1 h. Then the reaction mixture was concentrated at room temperature

using a rotary vacuum evaporator. Fractionation by silica gel flash chromatography was

conducted with a gradient of ethyl acetate to ethyl acetate:methanol (6:1). The fraction

containing vabicaserin nitrone was condensed and further purified on a silica gel TLC plate using

a developing solvent system of ethyl acetate:methanol (6:1). The yellow band containing

vabicaserin nitrone (Rf =0.3) fluoresced under UV (365 nm) irradiation. This band was extracted

using methanol and evaporated to dryness under a stream of nitrogen yielding vabicaserin

nitrone (0.3 mg). LC/MS data for the synthetic material matched that obtained for P5 in human

plasma (data not shown).

Incubation with Liver Microsomes. In vitro incubations were conducted to isolate metabolites

by HPLC for structure elucidation as well as to evaluate in vitro metabolite profiles in various

species. In vitro incubations with liver microsomes of mouse, rat, dog, monkey and human in

the presence of NADPH and UDPGA were the same as previously described (Tong et al. 2010).

Briefly, [14C]vabicaserin (10 μM) was incubated for 20 min in a CO2-enriched environment with

liver microsomes (0.5 mg/mL) in 0.5 mL of 50 mM bicarbonate buffer, pH 7.4, containing

alamethicin (50 μg/mg protein) and magnesium chloride (10 mM), in the presence of an NADPH

regenerating system and UDPGA (2 mM). In addition, [14C]vabicaserin (10 μM) was also

incubated for 20 min with liver microsomes (0.5 mg/mL) in 0.1 M phosphate buffer containing

magnesium chloride (10 mM), pH 7.4, in the presence of the NADPH regenerating system, in



SPET



Dow

nloaded from


8

order to study the oxidative metabolism alone. The samples were prepared and analyzed as

previously described (Tong et al. 2010) by HPLC with radioactivity detection for metabolite

profiles and by LC/MS for metabolite identification. Vabicaserin metabolites were isolated for

analysis by NMR spectroscopy.

Excretion Studies in Animals

All animal housing and care was conducted in Association for Assessment and Accreditation of

Laboratory Animal Care (AAALAC) accredited facilities. Animal care and use for excretion and

metabolism studies were approved by the Wyeth Institutional Animal Care and Use Committee.

Animal rooms were maintained on a 12-hour light and dark cycle. Animals were provided food

and water ad libitum.

Rats: Four male Sprague-Dawley rats, obtained from Charles River Laboratories (Wilmington,

MA), were used in this study. The animal body weights ranged from 337 to 348 g on the day of

dosing. The dose vehicle contained 2% Tween-80 and 0.5% methylcellulose in water.

Appropriate amounts of [14C]vabicaserin and non-labeled vabicaserin were dissolved in the dose

vehicle to give a solution. The final concentration of vabicaserin was approximately 2 mg/mL.

All animals (n=4) received a targeted single oral dose of 5 mg/kg (80.7 μCi/kg) via intragastric

gavage at a volume of 2.5 mL/kg. Urine and feces were collected for 120 h from all animals

following administration of [14C]vabicaserin. Urine samples were collected at ambient

temperature at intervals of 0-8, 8-24, 24-48, 48-72, 72-96, and 96-120 h post-dosing. Fecal

samples were collected at ambient temperature at intervals of 0-24, 24-48, 48-72, 72-96 and

96-120 h post-dosing. Cage rinses were collected at intervals of 0-24, 24-72 and 72-120 h by

rinsing each cage with approximately 100 mL of 30% ethanol in water.



SPET



Dow

nloaded from


9

Bile study in rats: Four male rats weighing from 387 to 411 g and four female rats weighing

from 291 to 325 g at the time of dosing were purchased from Charles River Laboratories

(Wilmington, MA), and bile-duct cannulation was performed in-house. The dose vehicle was the

same as in the excretion study. On the day of dosing, [14C]vabicaserin and non-labeled

vabicaserin were dissolved in the vehicle to a final concentration of approximately 1 mg/mL.

Non-fasted rats were given a single 5 mg/kg (323 μCi/kg) target dose of vabicaserin at a volume

of 5.0 mL/kg via intragastric gavage. Animals were provided standard rat chow and water ad

libitum, and were kept in metabolism cages individually.

Bile was collected into tubes on dry ice at 0-4, 4-8, 8-24 and 24-48 h intervals post-dosing.

Feces and urine were collected into containers on dry ice at 0-24 and 24-48 h intervals.

All biological samples and aliquots from the pre-dosing and post-dosing formulation were stored

at approximately -70°C until analyzed.

Dogs: Four male beagle dogs, weighing from 7.6 to 9.8 kg at the time of dosing, were from an

in-house colony. [14C]Vabicaserin hydrochloride and of non-labeled vabicaserin hydrochloride

were dissolved in methanol and then evaporated under a nitrogen stream to dryness. Capsules

(#2) were filled with accurate amounts (approximately 130 mg) of the mixed drug substance

according to animal weights to give a dosage of 15 mg/kg (39 μCi/kg). The filled gelatin

capsules were then enteric-coated manually. Each dog was given one enteric-coated capsule

containing [14C]vabicaserin as a hydrochloride salt. Animals were fed two hours prior to dosing

and provided Purina dog chow and water ad libitum, and were housed individually in metabolic

cages.



SPET



Dow

nloaded from


10

Urine samples were collected at intervals of 0-8, 8-24 and 24-48 h into tubes on dry ice, and at

intervals of 48-72, 72-96, 96-120, 120-144 and 144-168 h post-dosing at room temperature.

Fecal samples were collected at ambient temperature at intervals of 0-8, 8-24, 24-48, 48-72,

72-96, 96-120, 120-144 and 144-168 h post-dosing. Cage rinses were collected daily by rinsing

each cage with approximately 250-1100 mL of 30% ethanol in water.

In Vivo Metabolism

For metabolism studies in male mice, rats and dogs, radio-labeled doses were used. Male CD-1

mice and Sprague-Dawley rats were purchased from Charles River Laboratories (Wilmington,

MA). The dose vehicle for mice and rats contained 2% (w/w) Tween 80 and

0.5% methylcellulose in water. Twenty (five/time point) non-fasted male mice weighing from

28 to 34 grams at the time of dosing were given a single 50 mg/kg (~300 μCi/kg) dose of

vabicaserin at a volume of 20 mL/kg via intragastric gavage. Mice were kept in metabolic cages

in groups of five. Twelve (three/time point) non-fasted male rats weighing from approximately

320 to 350 grams at the time of dosing were given a single 5 mg/kg (~300 μCi/kg) dose of

vabicaserin at a volume of 2.5 mL/kg via intragastric gavage. Rats were kept individually in

metabolism cages. Dog plasma, urine and feces for metabolite profiling were collected from the

same animals used in the excretion study described earlier.

Four male cynomolgus monkeys, weighing from approximately 5 to 10 kg at the time of dosing,

were from an in-house colony. Non-fasted monkeys were given a single 25 mg/kg dose of non-

radiolabeled vabicaserin at a volume of 2 mL/kg via intragastric gavage. The vehicle was the

same as used in mice and rats. Animals were housed individually in metabolic cages.



SPET



Dow

nloaded from


11

Blood samples were collected from mice and rats at sacrifice by cardiac puncture at 2, 4, 8 and

24 h post-dosing. Blood samples of about 3 mL from the jugular vein of dogs and from the

femoral trigon of monkeys were collected at 2, 4, 8 and 24 h post-dosing. Potassium EDTA was

used as the anticoagulant and plasma was immediately harvested from the blood by

centrifugation at 4°C. Urine samples were collected from animals at 0-8 and 8-24 h intervals for

all species. Fecal samples were collected at 0-24 h post-dosing. Urine and feces were also

collected 24-48 h post-dose from dogs. All biological specimens were stored at approximately -

70°C until analysis.

Dosing and sample collections from humans were conducted at a single investigational site

(Methodist Hospital, Philadelphia, PA). Oral doses of vabicaserin capsules of 500 mg were

administered to three healthy male subjects under fasting conditions. Plasma samples were

collected within two hours before test article administration (pre-dose), and at 6, 12 and 24 h

post-dose for metabolite profiling. Urine specimens were collected at intervals of 0-4, 4-12 and

12-24 h. Samples were stored at approximately -70°C until analysis.

For human studies, the protocol and the informed consent forms (ICFs) were reviewed and

approved by the study site institutional review board (IRB). Subsequent amendments to the

protocols and/or any revisions to the ICFs were also reviewed and approved by the IRB. This

study was conducted in accordance with ethical principles that have origins in the Declaration of

Helsinki and in any amendments that were in place when the study was conducted. Written

informed consent was obtained from all subjects before their enrollment.

Radioactivity Determination for Mouse, Rat and Dog Studies. Plasma (10 μL) and urine

(100 μL) aliquots were analyzed for radioactivity concentrations. Each urine sample was mixed



SPET



Dow

nloaded from


12

and triplicate aliquots (100 or 200 μL) were analyzed for radioactivity concentrations. Prior to

analysis, cage rinses were weighed and mixed. Triplicate aliquots (100 or 200 μL) were weighed

and analyzed to determine the concentration of radioactivity. Radioactivity in plasma, urine and

cage rinses were determined with a Tri-Carb Model 3100 TR/LL liquid scintillation counter

(LSC) (PerkinElmer) using 5 mL of Ultima Gold as the scintillation fluid. Fecal samples were

weighed and homogenized in water at volume-to-weight ratios of about 5:1, and homogenates

(0.1-0.3 g) were placed on Combusto-cones with Combusto-pads and combusted. A model

307 Tri-Carb Sample Oxidizer, equipped with an Oximate-80 Robotic Automatic Sampler

(PerkinElmer), was used for combustion. The liberated 14CO2 was trapped with Carbo-Sorb E

carbon dioxide absorber, mixed with PermaFluor® E+ liquid scintillation cocktail, and counted in

a Tri-Carb Model 3100 TR/LL liquid scintillation counter (PerkinElmer). The oxidation

efficiency of the oxidizer was 97.7%.

Sample Preparation for Metabolite Profiling and Metabolite Identification. Plasma samples

were pooled by mixing an equal volume from each animal for each time point, and processed as

previously described (Tong et al., 2010). Briefly, aliquots of pooled plasma were mixed with

two volumes of methanol, placed on ice for about 5 min and then centrifuged. The supernatant

was transferred to a clean tube and evaporated at 22°C under nitrogen in a TurboVap LV

(Caliper Life Sciences) to a volume of about 0.3 mL. The concentrated extracts were

centrifuged, the supernatant volumes measured, and duplicate 10 μL aliquots analyzed by LSC

for extraction efficiency. Extraction efficiency was determined by comparing the total

radioactivity in the extracts versus that in the samples prior to extraction. An average of greater

than 80% of the plasma radioactivity was recovered in the extracts. The 24 h plasma samples

from mice, rats and dogs were not analyzed due to low radioactivity concentration. Urine



SPET



Dow

nloaded from


13

samples were pooled in a proportional manner to give 0-24 h samples for mouse, rat and dog.

Pooled urine samples were analyzed without further preparation.

Fecal homogenates were pooled proportionally to their weight to give 0-24 h samples for mouse,

rat and dog. Aliquots of 1 g of the pooled fecal homogenate were mixed with 2 mL methanol,

placed on ice for about 10 min and centrifuged. The supernatant was transferred to a clean tube.

The residue was extracted three times with 2 mL of a water:methanol (3:7) mixture. The

supernatants from each sample were combined, evaporated to about 1 mL, and centrifuged.

Extraction efficiency was determined by analyzing aliquots of 10 μL of the supernatant, by

comparing the total radioactivity in the extracts versus the total radioactivity in the samples prior

to extraction. An average of greater than 70% of the fecal radioactivity was extracted. Monkey

and human fecal samples were not analyzed.

Aliquots of urine and extracts of plasma and fecal samples were analyzed by HPLC with

radioactivity detection described below for metabolite profiles and by LC/MS for metabolite

characterization.

High Performance Liquid Chromatography (HPLC). A Waters model 2695 HPLC system

(Waters Corp., Milford, MA) with a built-in autosampler was used for metabolite isolation and

analysis for metabolite profiles. Separations were accomplished on a Luna C18(2) column

(150 x 2.0 mm, 5 μm) (Phenomenex, Torrance, CA) coupled with a guard (4 x 2 mm) cartridge.

The sample chamber of the autosampler was maintained at 4°C, while the column was at an

ambient temperature of about 20°C. Radioactivity chromatograms were recorded as described

above. The mobile phase consisted of 10 mM ammonium acetate, pH 4.5 (A) and methanol (B)

and was delivered at 0.2 mL/min. The linear gradients used are listed in Table 2 for each study.



SPET



Dow

nloaded from


14

The HPLC eluent was collected at 20 s intervals into 96-well Lumaplates (PerkinElmer) due to

low radioactivity concentrations in mouse and dog plasma samples. The plates were dried

overnight in an oven at 40°C and analyzed by the TopCount NXT radiometric microplate reader.

For other radioactive animal samples and in vitro incubations, a Flo-One β Model A525

radioactivity detector with a 250 μL flow cell was used for data acquisition. The flow rate of

Ultima Flow M scintillation fluid was 1 mL/min, providing a mixing ratio of scintillation

cocktail to mobile phase of 5:1.

Liquid Chromatography/Mass Spectrometry (LC/MS). For metabolite characterization, the

HPLC conditions were the same as described above, except that Agilent model 1100 (Agilent

Technologies, Palo Alto, CA), Waters 2695 and Acquity UPLC (Waters) liquid chromatography

systems were used. UV spectra were recorded with diode array UV detectors for all analyses.

Most of the LC/MS analyses were conducted with mobile phase Gradient C (Table 2). Mass

spectral data for vabicaserin and its metabolites were obtained with LCQ ion trap (Thermo, San

Jose, CA), quadrupole time-of-flight (Q-TOF) (Waters) and triple quadrupole (Waters) mass

spectrometers. Each mass spectrometer was equipped with an electrospray ionization source and

operated in the positive ionization mode. Settings for each mass spectrometer were optimized to

provide a structurally relevant range of product ions from MS/MS and MSn experiments.

Product ion spectra recorded with the triple quadrupole mass spectrometer employing a collision

energy setting of 22 eV were used as the reference spectra to confirm metabolite identifications.

MassLynx (versions 3.5, 4.0, and 4.1, Waters) and Xcalibur (version 1.3, Thermo) software were

used for collection and analysis of LC/MS data.



SPET



Dow

nloaded from


15

Nuclear Magnetic Resonance (NMR) Spectroscopy. Vabicaserin metabolites P1, P2, P3, P4,

P5, P6 and CG were isolated, concentrated and re-purified using the same HPLC conditions as

above except that 0.1% formic acid in water was used as mobile phase A and 0.1% formic acid

in methanol was used as mobile phase B. Vabicaserin metabolites P7, P9 and P10 were isolated

from rat urine by HPLC with an ammonium acetate mobile phase followed by HPLC with a

trifluoroacetic acid mobile phase. Each isolated metabolite was evaporated to dryness under a

nitrogen stream. Vabicaserin metabolites P1, P2, P3, P4, P5 and P6 were dissolved in 500 μL

DMSO-d6 and transferred into a 5 mm NMR tube. NMR spectroscopic data were recorded at

30ºC on a 600 MHz Bruker Avance III spectrometer with a 5 mm CPTCI CryoProbe™ (Bruker

BioSpin Corporation, Billerica, MA). Proton, HSQC, and HMBC experiments were performed.

All chemical shifts were referenced to the DMSO signal at 1H δ 2.49 ppm and 13C δ 39.5 ppm.

The proton spectra were acquired with 32,768 data points over a 6,602 Hz spectral window using

a 30º pulse and standard pulse sequence. Data were Fourier transformed with a 0.3 Hz line

broadening window function. The 2D 1H-13C HSQC spectra were acquired with 1024 data

points in F2, and 256 increments in F1, with 32 scans per increment, using a phase sensitive pulse

sequence. The 2D 1H-13C HMBC spectra were acquired with 4096 data points in F2, and 256

increments in F1 with 64 scans per increment. Vabicaserin metabolites P7, P9 and P10 were

dissolved in 150 μL DMSO-d6 and transferred into a 3 mm NMR tube. One-dimensional (1D)

proton NMR and two-dimensional (2D) NMR (COSY, ROESY) data were collected on an Inova

500 MHz NMR spectrometer (Varian, Palo Alto, CA) equipped with a Nalorac 5 mm z-gradient

indirect detection probe (Varian).



SPET



Dow

nloaded from


16

Results

Metabolite Characterization. Mass spectral data for vabicaserin and its metabolites are

summarized in Table 3. Based on their molecular ions and fragmentations in comparison to

vabicaserin, the proposed structures of metabolites are presented in Figure 1. Vabicaserin

metabolites were isolated by HPLC from in vitro incubations (P1, P2, P3, P4, P5, P6 and CG)

and from rat urine (P7, P9 and P10) for analysis by LC-MS/MS and NMR spectroscopy, and the

structures of the aforementioned metabolites were confirmed by NMR spectroscopic data from

1H and 13C (1H-1H COSY, 1H-13C HSQC, and 1H-13C HMBC) (Table 4). HPLC retention time

and mass spectral data for CG matched those for the synthetic carbamoyl glucuronide of

vabicaserin.

In Vitro Metabolite Profiles

Radiochromatographic profiles of [14C]vabicaserin (10 μM) following incubation with human

liver microsomes in the absence and presence of the NADPH regenerating system, and in the

presence of both NADPH and UDPGA are depicted in Figure 2. When [14C]vabicaserin was

incubated with human liver microsomes in phosphate buffer in the presence of NADPH, five

metabolites (P1-P5) were detected. Formation of P6 required microsomes, but was not NADPH-

dependent. Metabolites P1, P2, P3 and P4 were characterized by LC/MS and NMR spectroscopy

as hydroxyl vabicaserin, P5 as vabicaserin nitrone and P6 as vabicaserin imine. When

[14C]vabicaserin was incubated with human liver microsomes in bicarbonate buffer and a CO2-

enriched environment in the presence of both NADPH and UDPGA, a new product (CG) was

detected with higher abundance than any of the other aforementioned metabolites. CG was

identified as vabicaserin carbamoyl glucuronide by LC/MS analysis and comparison with the

synthetic carbamoyl glucuronide.



SPET



Dow

nloaded from


17

Radiochromatographic profiles of [14C]vabicaserin following incubation at a concentration of 10

μM with mouse, rat, dog and monkey liver microsomes in presence of both NADPH and

UDPGA are shown in Figure 3. Although quantitative differences among the various species

investigated were apparent, the in vitro metabolite profiles of vabicaserin were qualitatively

similar across the species (Figures 2 and 3), and all human metabolites were detected in one or

more animal species. Monkey liver microsomes generated similar profiles to those of humans,

except that P2 was detected in relatively higher intensity, while P6 was observed in relatively

lower intensity than in human liver microsomes. However, as the purpose of the in vitro study

was to generate metabolites for structural elucidation and for qualitative profiling, the in vitro

incubation conditions were not optimized for each species to provide in vitro and in vivo

extrapolation.

Excretion

Mice: Excretion of radioactivity was determined in the samples collected up to 24 h following

oral administration of 50 mg/kg [14C]vabicaserin to male CD-1 mice in the metabolism study.

Urine was the major route of excretion, with 59.6% of the radioactive dose recovered in the 24 h

post-dose. Fecal elimination in the first 24 h post-dose accounted for 13.6% of the dosed

radioactivity.

Rats: The excretion of radioactivity in male Sprague-Dawley rats was evaluated after a single

oral (gavage) dosage of 5 mg/kg [14C]vabicaserin. The total recovery of radioactivity (feces,

urine, and cage rinse) over a period of 5 days (120 h) was 93.8%. The major route of excretion

of radioactivity was the urine (64.3%); fecal excretion was relatively minor (28.0%). The rate of

excretion of radioactivity was rapid, with 87.4% of the oral dose recovered in urine and feces

(including cage rinse) in the first 24 h after dosing.



SPET



Dow

nloaded from


18

Biliary excretion in rats: Excretion of radioactivity was also determined in bile-duct cannulated

rats after a single oral 5 mg/kg dose of [14C]vabicaserin. Urine was the major excretion route for

vabicaserin related radioactivity after a single oral dose, while biliary excretion was relatively

minor. In the first 48 h post-dosing, an average of 60% of the radioactive dose was excreted in

the urine, while an average of 17% of the administered radioactivity was excreted in the bile.

Excretion of radioactivity in rats was rapid, with the majority of the radioactivity recovered in

the first 24 h post-dosing. These results were consistent with the finding in the mass balance

study, where 64% of the administered radioactivity was recovered in urine.

Dogs: The excretion of radioactivity in male beagle dogs was evaluated after a single oral

(enteric-coated capsule) dose of 15 mg/kg of [14C]vabicaserin. The total recovery of

radioactivity (feces, urine, and cage rinse) over a period of 7 days (168 h) was 97.3%. Large

individual variations in daily recovery were observed. The major route of excretion of

radioactivity was the feces (58.5%); urinary excretion was relatively minor (32.7%). The rate of

excretion of radioactivity was rapid, with 84.2% of the oral dose recovered in urine and feces

(including cage rinse) in the first 48 h after dosing, which may have been due to the enteric

coating affecting the release of vabicaserin.

In Vivo Metabolite Profiles

Representative radiochromatographic profiles of plasma and urine samples of mice, rats and

dogs administered a single oral dose of [14C]vabicaserin are depicted in Figures 4-6. Vabicaserin

was extensively metabolized in all three species, with the parent drug representing less than 21%

of the total plasma radioactivity. Estimated concentrations of vabicaserin and its metabolites

observed in plasma of mice, rats and dogs following oral administration of [14C]vabicaserin are



SPET



Dow

nloaded from


19

summarized in Table 5. Percentages of vabicaserin and its metabolites in urine of various

species as determined by their chromatographic distribution are summarized in Table 6.

In mice, vabicaserin, CG and hydroxyl vabicaserin (P2 and P3) were the major radioactive

components in plasma (Figure 4). Vabicaserin represented no more than 1.2% of the

administered radioactive dose in urine. Hydroxyl vabicaserin (P2 and P3), hydroxyl vabicaserin

glucuronide (P9), didehydrohydroxyl vabicaserin glucuronide (P19), and CG were the major

metabolites in mouse urine. Unchanged vabicaserin represented 18% of fecal radioactivity in 0

to 8 h post-dose and 11% for 8 to 24 h post-dose samples. Hydroxyl vabicaserin (P2 and P3),

keto vabicaserin (P7), hydroxyl vabicaserin imine sulfate (P8), hydroxyl vabicaserin sulfate

(P10), and hydroxyl vabicaserin imine (P14) were identified in feces. CG was not observed in

mice feces, likely because of cleavage in the gastrointestinal tract.

In rats, vabicaserin was extensively metabolized to predominantly oxidative metabolites.

Unchanged vabicaserin represented ≤ 20% of the total radioactivity in plasma and < 1% of the

administered radioactive dose in urine. Hydroxyl vabicaserin (P1, P2 and P3) and keto

vabicaserin (P7) were the major drug-related components in rat plasma (Figure 5). The hydroxyl

metabolites (P1 and P3), keto vabicaserin (P7), and the glucuronide (P9) were the major

metabolites in urine. CG was not detected in rat plasma or urine. Metabolites P3, P8, P10, and

P11 and only trace amounts of vabicaserin were detected in rat feces.

In the biliary excretion and metabolite profile study in rats, vabicaserin represented an average of

< 3% of radioactivity in bile, and 3% to 4% of radioactivity in urine. Vabicaserin and its

metabolites were excreted in bile almost solely as conjugates. Biliary metabolite profiles did not

change significantly over time. The major biliary metabolites included CG, hydroxyl vabicaserin



SPET



Dow

nloaded from


20

imine sulfate (P8), hydroxyl vabicaserin glucuronides (P9 and P24), N-acetyl hydroxyl

vabicaserin (P12), and hydroxyl vabicaserin carbamoyl glucuronide (P21). CG, which was not

detected in plasma or urine, was the most abundant metabolite in bile, representing 22% to 30%

of the radioactivity in bile. Urinary metabolites included hydroxyl vabicaserin (P1, P2, P3, and

P4), keto vabicaserin (P7), hydroxyl vabicaserin imine sulfate (P8), hydroxyl vabicaserin sulfate

(P10), hydroxyl vabicaserin glucuronide (P9), and N-acetyl hydroxyl vabicaserin (P12).

In dogs, vabicaserin was extensively metabolized after administration of an enteric-coated

capsule containing [14C]vabicaserin. Oxidative metabolism was the major metabolic pathway,

while formation of CG was also observed. Vabicaserin represented < 21% of the radioactivity in

plasma and no more than 0.5% of the administered radioactive dose in urine, similar to mice and

rats. In dog plasma, vabicaserin, CG, P1, P2, P3 as well as hydroxyl vabicaserin imine (P13)

were the major drug-related components. Metabolites observed in dog plasma were also detected

in dog urine. A hydroxyl vabicaserin sulfate (P17) and a diazepinyl vabicaserin carboxylic acid

(P18), which was not detected in plasma, were observed in urine. Hydroxyl vabicaserin

metabolites (P2, P3, and P20), a keto vabicaserin (P7), and a hydroxyl vabicaserin imine (M15)

were detected in fecal extracts. Some metabolites of vabicaserin differed in dogs compared with

those in rats. Sulfate metabolites were less abundant in dogs, and CG was detected in dogs but

not in rats.

Representative summed mass chromatograms of plasma and urine samples obtained from

monkeys and humans following oral administration vabicaserin are shown in Figures 7 and 8.

Non-labeled material was administered to monkeys and humans, and oxidative metabolites were

not quantified in these samples. In monkeys, vabicaserin was extensively metabolized via both

phase I and II metabolism. In monkey plasma, along with the metabolites observed in the in



SPET



Dow

nloaded from


21

vitro incubation with monkey liver microsomes, additional oxidative metabolites (P20 and P25)

were present (Figure 7).

After oral (capsule) administration of vabicaserin to humans, vabicaserin was extensively

metabolized, primarily to the carbamoyl glucuronide (CG). Vabicaserin nitrone (P5) was

observed in humans, as seen in dog plasma (Figures 6 and 8). Generally, urinary metabolite

profiles were similar to plasma profiles (Figures 7-8). A number of metabolites, which were not

detected in in vitro incubations, were observed in plasma and/or urine of humans and animal

species examined. These were the secondary metabolite formed through oxidative metabolism

and conjugation of the primary metabolites of hydroxylation, imine formation or carbamoyl

glucuronidation.



SPET



Dow

nloaded from


22

Discussion

The present study indicated that vabicaserin undergoes NADPH-dependent oxidative metabolism

to form hydroxyl metabolites (P1-P4), NADPH-independent formation of an imine (P6) as well

as UGT-catalyzed formation of a carbamoyl glucuronide (CG) in a CO2 enriched environment in

human liver preparations (Figure 9). Vabicaserin nitrone (P5) was generated directly from

vabicaserin or from P6 in human liver microsomes in the presence of NADPH.

Radioactivity was eliminated rapidly in mice (73.2% in 24 h), rats (87.4% in 24 h) and dogs

(84.2% in 48 h) following oral administration of [14C]vabicaserin. Urine was the major route of

elimination of the dosed radioactivity following oral administration of [14C]vabicaserin to mice

and rats, while fecal elimination was the major route of excretion in dogs. Since non-

radiolabeled vabicaserin was administered to monkeys and humans, excretion of total drug

derived materials could not be determined. However, quantitative data obtained by LC/MS

analysis indicated that less than 1% of the oral dose was eliminated as unchanged drug and >50%

of the administered dose was excreted as CG in urine of humans (Tong et al 2010).

Vabicaserin was extensively metabolized in all species and species differences were observed

following a single oral dose to humans and animals. Although a number of additional

metabolites were observed, in vivo metabolism of vabicaserin generally followed the same

metabolic pathways observed in vitro: hydroxylation, imine formation and carbamoyl

glucuronidation. Further metabolism of the primary metabolites through oxidative metabolism

and conjugation yielded additional metabolites. As previously discussed (Tong et al., 2010),

carbamoyl glucuronidation was the major metabolic pathway in humans, but a minor metabolic

pathway in rats. Carbamoyl glucuronidation was also a major metabolic pathway in mice, dogs



SPET



Dow

nloaded from


23

and monkeys. However, oxidative metabolism was also extensive in these species.

Hydroxylation reactions occurred in all species, although different regional selectivity seemed to

exist. Although vabicaserin imine was observed only in humans, secondary metabolites of the

imine were observed in all species except monkey, suggesting that this metabolic pathway also

exists in mice, rats and dogs. Vabicaserin nitrone (P5) was observed in dogs and humans but not

in the other species studied.

Vabicaserin is composed of a fused four-ring system, including benzene, diazepane, piperidine,

and cyclopentane rings. Few metabolites were observed as a result of oxidation on the benzene

ring or at positions of 6, 7, 8, and 9 (Figure 1) even though these positions were considered an

electron rich region. Instead, oxidation occurred either on the cyclopentane ring with the

benzene and diazepane rings serving as the metabolic enzyme binding region to generate P1 to

P4, or on the diazepane ring with the other side of the molecule as the binding region for P450 or

other oxidation enzymes to form P5 and P6. Identification of enzymes responsible for

generating these metabolites is under investigation; as such information would be critical to

determine potential clinical drug-drug interactions.

In conclusion, vabicaserin was extensively metabolized in mice, rats, dogs, monkeys and humans

following oral administration. Urinary excretion was the major route of elimination of the dosed

radioactivity following oral administration of [14C]vabicaserin to mice and rats, while fecal

elimination was the major route of excretion in dogs. The structures of the metabolites isolated

and identified by LC/MS and NMR spectroscopic analysis from in vitro incubation of

vabicaserin with human liver microsomes indicated that vabicaserin metabolism involved three

major metabolic pathways: NADPH-dependent hydroxylation, NADPH-independent formation

of vabicaserin imine and carbamoyl glucuronidation.



SPET



Dow

nloaded from


24

Acknowledgment

The authors thank Hongshan Li, Michael Carbonaro, C. Paul Wang, Abdul Mutlib, Theresa

Hultin and Rasmy Talaat for their contributions to these studies.



SPET



Dow

nloaded from


25

References

Delbressine L, Funke C, van Tilborg M and Kaspersen F (1990) On the formation of carbamate glucuronides. Xenobiotica 20:133-134.

DiGiovanni G, DiMatteo V, DiMascio M and Esposito E (2000) Preferential modulation of mesolimbic vs nigrostriatal dopaminergic function by serotonin 2C/2B receptor antagonists: a combined in vivo electrophysiological and microdialysis study. Synapse 35:53-61.

DiMatteo V, DiGiovanni G, DiMascio M and Esposito E (1999) SB 242084, a selective serotonin 2C receptor antagonist, increases dopaminergic transmission in the mesolimbic system. Neuropharmacology 38:1195-1205.

Dunlop J, Zhang J, Watts S, Ramamoorthy S, Harrison B, Barrett JE, Magolda R, Schecheter L, Pangalos MN, Stack G and Rosenzweig-Lipson S (2006) In vitro pharmacological profile of SCA-136, a novel 5-HT2C receptor selective agonist. 36th Annual Meeting for the Society for Neuroscience (Washington, DC), Atlanta, GA, Oct 14-18.

Fenselau C (1994) Acyl glucuronides as chemically reactive intermediates. Hdbk Exp Pharmacol 112:367-389. Gipple K, Tung C, Elvin A, Lalka D and Alexon J (1982) Species differences in the urinary excretion of the novel

primary amine conjugate: tocainide carbamoyl-O-β-D-glucuronide. J Pharm Sci 71:1011-1014. Greenaway W and Whatley F (1987) Formation of N-carboxy compounds during trimethylsilanization of amino

acids in the presence of carbon dioxide. J Chromatography 409:383-389. Kilmartin J and Rossi-Bernardi L (1973) Interaction of hemoglobin with hydrogen ions, carbon dioxide and organic

phosphates. Physiol Rev 53:83-890. Marquis KL, Dunlop J, Ramamoorthy S, Beyer CE, Lin Q, Brennan J, Piesla MJ, Ashby CRJ, Harrison B, Magolda

R, Pangalos MN, Stack G and Rosenzweig-Lipson S (2006) SCA-136: a novel 5-HT2C receptor agonist possessing atypical antipsychotic-like effects in preclinical models. 36th Annual Meeting for the Society for Neuroscience (Washington, DC), Atlanta, GA, Oct 14-18.

Millan M, Dekeyne A and Gobert A (1998) Serotonin (5-HT)2C receptors tonically inhibit dopamine (DA) and noradrenaline (NA), but not 5-HT, release in the frontal cortex in vivo. Neuropharmacology 37:953-955.

Morrow J, Keim P and Gurd F (1974) CO2 adducts of certain amino acids, peptides and whale myoglobin studied by carbon 13 and proton nuclear magnetic resonance. J Biol Chem 249:7484-7494.

Mroz E (1989) Possible role of carbamates in neurotoxicity and neurotransmitter inactivation. Science 243:1615. Schaefer W (1992) Formation of a carbamoyl glucuronide conjugate of carvedilol in vitro using dog and rat liver

microsomes. Drug Metabolism and Disposition 20:130-133. Schaefer W (2006) Reaction of primary and secondary amines to form carbamic acid glucuronides. Curr Drug

Metab 7:873-881. Senda C, Toda S, Tateishi M, Kobayashi K, Igarashi T and Chiba K (2003) Mexiletine carbonyloxy b-D-

glucuronide: a novel metabolite in human urine. Xenobiotica 8:871-884. Spahn-Langguth H and Benet L (1992) Acyl glucuronides revisited: is the glucuronidation process a toxification as

well as a detoxifixation mechanism? Drug Metab Rev. 24:5-47. Tremaine L, Stroh J and Ronfeld R (1989) Characterization of a carbamic acid ester glucuronide of the secondary

amine sertraline. Drug Metab Dispos 17:58-63. Tong Z, Chandrasekaran A, DeMaio W, Jordan R, Li H, Moore R, Poola N, Burghart P, Hultin T and Scatina J

(2010) Species differences in the fromation of vabicaserin carbamoyl glucuronide. Drug Metab Dispos 38:1-10.

Weiss J and Choi D (1988) Beta-N-methylamino-L-alanine neurotoxicity: requirements for bicarbonate as cofactor. Science 241:973-975.



SPET



Dow

nloaded from


26

Footnotes

Address correspondence to: Dr. William DeMaio, 4 Wynnewood Dr, Collegeville, PA 19426. E-

mail: [email protected]



SPET



Dow

nloaded from


27

Figure Legends Figure 1. Structures of vabicaserin and its metabolites

Figure 2. Incubations of [14C]vabicaserin (10 μM) with human liver microsomes in the absence

(A) and presence (B) of the NADPH regenerating system, and in the presence of both

NADPH and UDPGA (C)

Figure 3. Incubations of [14C]vabicaserin (10 μM) with liver microsomes in presence of both

NADPH and UDPGA

Figure 4. Radiochromatograms of Mouse Plasma (2 h) and Urine (0-24 h)

Figure 5. Radiochromatograms of Rat Plasma (2 h) and Urine (0-24 h)

Figure 6. Radiochromatograms of Dog Plasma (4 h) and Urine (0-24 h)

Figure 7. Summed Mass Chromatogram of Vabicaserin and its Metabolites in Pooled Monkey

Plasma (2 h) and Urine (0-24 h)

Figure 8. Summed Mass Chromatogram of Vabicaserin and its Metabolites in Human Plasma (6

h) and Urine (0-24 h)

Figure 9. In Vitro Metabolic Pathways of Vabicaserin in Humans



SPET



Dow

nloaded from


28

Table 1. Characteristics of mouse, rat, dog, monkey and human liver microsomes

utilized in this study

Species Sex

Number of Subjects

Pooled

P450 Content

(nmol/mg protein)

Mouse Male 20 0.40

Female 18 0.54

Rat Male 23 0.79

Female 50 0.55

Dog Male 5 0.57

Female 4 0.43

Monkey Male 10 1.17

Female 9 1.31

Human Male and female

mixed

10 0.36



SPET



Dow

nloaded from


29

Table 2. LC Gradients used for HPLC and LC/MS Analysis

Gradient A for analysis of in vitro samples Time (min) A (%) B (%)

0 90 10 3 90 10

25 60 40 45 15 85 50 15 85

Gradient B for analysis of rat and dog samples Time (min) A (%) B (%)

0 90 10 6 90 10

35 60 40 65 15 85

Gradient C for analysis of mouse, monkey and human samples Time (min) A (%) B (%)

0 95 5 3.0 95 5 3.1 90 10

13.0 90 10 25 80 20 50 70 30 51 50 50 70 10 90 80 10 90



SPET



Dow

nloaded from


30

Table 3. Mass spectral data for vabicaserin and its metabolites

Metabolite [M+H]+

Species/Matrixa

Relevant Product Ions (m/z) Mouse Rat Dog Monkey Human

Vabicaserin 229 P, U, F P, U, F, B P, U P, U P, U 212, 200, 187, 186, 158, 144, 132 P1 245 P, U P, U P, U P, U 228, 227, 216, 202, 186, 184, 158, 144 P2 245 P, U, F P, U P, U, F 228, 216, 202, 198, 184, 158, 144, 132 P3 245 P, U, F P, U, F P, U, F P P, U 228, 227, 216, 202, 198, 184, 158, 144, 132 P4 245 P, U 227, 198, 184, 156 P5 243 P, U P 226, 225, 198, 157, 130 P6 227 P, U 210, 198, 184, 156, 130 P7 243 P, U, F P, U P, U, F 226, 214, 200, 198, 158, 144, 132, 98 P8 325 P, U, F, B 283, 202, 184, 158, 144, 132 P9 421 P, U P, U, B P, U P, U 378, 245, 203, 202, 187, 160

P10 325 F P, U, F 283, 202, 184, 158, 144, 132 P11 245 P, U, F 186, 171, 158, 144 P12 287 P, U, B 269, 227, 186, 171, 158, 144 P13 243 P, U 187, 169, 130 P14 243 P, U, F P, U 226, 214, 200, 158, 144, 132, 98 P15 243 F 214, 200, 196, 182, 130 P16 243 P, U 214, 200, 196, 182, 130 P17 325 U 283, 282, 202, 184, 148 P18 257 U 213, 186, 171, 145, 130 P19 419 U P, U 243, 214, 202, 187 P20 245 F P, U P, U 228, 216, 186, 171 P21 465 B U 421, 289, 245, 228, 202, 184, 144 P22 421 U 404, 386, 362, 269, 228, 227, 210, 187 P23 421 U 360, 271, 245, 229, 228, 227, 212, 210, 200, 186 P24 421 B P, U 404, 378, 245, 228, 227, 202 P25 243 P, U 226, 214, 200, 132 CG 449 P B P, U P, U P, U 273, 229, 212, 186, 113

a. P, plasma; U, urine; F, feces; B, bile. Fecal samples from monkeys and humans were not analyzed for metabolite profiles.

DMD # 33670T

his article has not been copyedited and formatted. T

he final version may differ from

this version.D

MD

Fast Forward. Published on A

ugust 25, 2010 as DO

I: 10.1124/dmd.110.033670

at ASPET Journals on August 4, 2018 dmd.aspetjournals.org Downloaded from


31

Table 4. NMR spectroscopic data for vabicaserin and its metabolites

Metabolite 1H NMR (600 or 500 MHz, DMSO-d6) δ ppm 13C NMR δ ppm Vabicaserin 9.77 (1H, br. s.), 8.81 (1H, br. s.), 7.22 (1H, d, J=7.45 Hz), 7.16 (1H, d, J=7.45 Hz), 6.90 (1H, t, J=7.45 Hz),

4.18 (1H, d, J=13.70 Hz), 4.04 (1H, d, J=13.70 Hz), 3.39 (1H, dd, J=11.59, 4.33 Hz), 3.13 (3H, m), 3.04 (1H, dd, J=12.89, 4.43 Hz), 2.93 (1H, ddd, J=10.98, 7.56, 7.45 Hz), 2.64 (1H, t, J=12.89 Hz), 2.23 (1H, ddd,

J=11.99, 8.06, 3.73 Hz), 2.18 (1H, m), 1.99 (1H, td, J=12.89, 9.77 Hz), 1.64 (1H, m), 1.54 (1H, m, J=10.23, 10.10, 10.10, 2.42 Hz), 1.33 (1H, dd, J=11.89, 7.86 Hz), 1.25 (1H, m)

147.07, 131.57, 130.85, 128.66, 124.73, 121.03, 55.44,

51.94, 48.95, 46.98, 40.79, 34.1, 31.42, 28.81, 23.31

P1 7.06 (1H, dd), 7.01 (1H, dd, J=6.75, 0.50 Hz), 6.81 (1H, t, J=7.45 Hz), 4.67 (1H, d, J=4.84 Hz), 4.15 (1H, t, J=8.06 Hz), 3.92 (1H, m), 3.85 (1H, m), 3.18 (1H, d, J=13.50 Hz), 3.05 (1H, m), 2.95 (1H, m), 2.89 (1H, m), 2.83 (1H, t, J=12.59 Hz), 2.37 (1H, dd, J=6.65, 5.24 Hz), 2.19 (2H, m), 1.35 (1H, td, J=12.14, 8.97 Hz), 1.08

(1H, ddd, J=12.54, 6.60, 2.82 Hz).

147.15, 131.15, 130.03, 129.12, 127.03, 120.50, 70.19,

56.56, 54.89, 51.30, 48.99, 42.67, 38.12, 37.76, 30.50

P2 7.07 (1H, d, J=7.66 Hz), 6.98 (1H, d, J=7.45 Hz), 6.81 (1H, t, J=7.35 Hz), 4.53 (1H, d, J=1.01 Hz), 4.10 (1H, dd, J=10.88, 5.24 Hz), 3.89 (1H, d, J=14.51 Hz), 3.81 (1H, d, J=13.70 Hz), 3.39 (1H, d, J=8.46 Hz), 3.18 (1H,

d, J=1.41 Hz), 3.02 (1H, d, J=3.83 Hz), 2.99 (1H, m), 2.92 (2H, m), 2.55 (1H, t, J=12.89 Hz), 2.32 (1H, tt, J=8.26, 4.03 Hz), 2.09 (1H, dd, J=13.20, 6.95 Hz), 1.89 (1H, dd, J=14.51, 7.05 Hz), 1.46 (2H, m)

147.33, 131.37, 130.38, 128.96, 126.79, 120.49, 69.56,

56.06, 55.24, 51.66, 49.33, 43.03, 39.29, 37.71, 30.19

P3 8.21 (1H, br. s.), 7.05 (1H, d, J=7.45 Hz), 6.94 (1H, d, J=7.05 Hz), 6.78 (1H, t, J=7.45 Hz), 4.71 (1H, br. s.), 3.81 (1H, br. s.), 3.81 (1H, d, J=12.49 Hz), 3.77 (1H, d, J=13.30 Hz), 3.18 (1H, m), 3.12 (1H, d, J=9.47 Hz), 3.08 (1H, dd, J=13.20, 4.13 Hz), 2.99 (1H, m), 2.91 (1H, m, J=10.48, 10.48, 10.38, 10.17 Hz), 2.61 (1H, t,

J=12.59 Hz), 2.27 (1H, ddd, J=8.21, 3.98, 3.73 Hz), 2.03 (1H, m), 1.90 (1H, m), 1.48 (1H, dddd, J=17.73, 8.16, 4.73, 4.63 Hz), 1.35 (1H, m, J=12.95, 9.37, 9.09, 9.09 Hz)

128.74, 126.62, 120.46, 75.41, 56.21, 53.4, 52.11, 49.71, 41.96, 38.27, 33.36, 32.19

P4 7.36 (1H, dt, J=7.66, 0.91 Hz), 6.98 (1H, dd, J=6.35, 0.91 Hz), 6.81 (1H, t, J=7.45 Hz), 4.73 (1H, s), 3.81 (1H, d, J=14.10 Hz), 3.78 (1H, d, J=13.70 Hz), 3.16 (1H, dd, J=2.82, 1.41 Hz), 3.04 (1H, m), 2.99 (1H, dd, J=13.80, 4.73 Hz), 2.91 (1H, t, J=9.87 Hz), 2.81 (1H, dd, J=13.70, 11.89 Hz), 2.16 (1H, dd, J=11.59, 3.53 Hz), 2.04 (1H,

m), 1.98 (1H, ddd, J=12.59, 8.26, 1.91 Hz), 1.76 (1H, m), 1.68 (1H, dd, J=12.59, 8.76 Hz), 1.52 (1H, ddd, J=8.21, 3.98, 3.73 Hz), 1.21 (1H, m)

146.52, 134.92, 130.96, 127.68, 127.15, 120.29, 78.39,

57.69, 56.45, 52.00, 49.42, 40.56, 40.03, 27.36, 21.02

P5 7.71 (1H, s), 7.06 (1H, d, J=7.25 Hz), 7.04 (1H, d, J=7.66 Hz), 6.67 (1H, t, J=7.56 Hz), 4.12 (1H, dt, J=15.72, 4.63 Hz), 4.02 (1H, dt, J=15.72, 4.43 Hz), 3.50 (2H, m), 3.12 (1H, dd, J=12.09, 5.24 Hz), 2.94 (1H, dd,

J=17.73, 7.66 Hz), 2.83 (1H, dd, J=11.89, 10.48 Hz), 2.28 (1H, m), 2.13 (1H, m), 1.92 (1H, m), 1.62 (1H, m, J=16.22, 8.11, 8.11, 4.43 Hz), 1.52 (1H, dddd, J=19.90, 10.17, 10.02, 3.02 Hz), 1.42 (1H, m), 1.36 (1H, m)

144.39, 136.92, 130.05, 130.05, 128.83, 117.4, 115.35,

64.93, 53.15, 51.22, 40.49, 34.61, 34.47, 28.67, 22.34

P6 8.66 (1H, s), 7.58 (1H, dd, J=8.16, 1.31 Hz), 7.47 (1H, d, J=7.05 Hz), 6.81 (1H, dd, J=7.96, 7.15 Hz), 3.94 (1H, m), 3.86 (1H, m), 3.59 (2H, d, J=3.63 Hz), 3.40 (1H, dd, J=5.04, 2.62 Hz), 3.16 (1H, d, J=1.81 Hz), 3.02 (1H, m), 2.31 (1H, m), 2.11 (1H, dddd), 1.93 (1H, dddd, J=12.87, 5.54, 5.36, 1.81 Hz), 1.66 (1H, m), 1.59 (1H, m),

1.46 (1H, dd, J=12.49, 10.48 Hz), 1.41 (1H, m)

164.67, 150.06, 137.87, 137.03, 128.61, 116.78,

111.18, 54.11, 52.95, 51.28, 40.46, 33.87, 33.74, 27.97,

21.59 P7 9.10, 8.62, 7.35, 7.23, 7.01, 4.18, 4.15, 3.47, 3.34, 3.16 (dd), 3.20, 3.06, 3.01 (t), 2.59, 2.53, 2.32, 2.14, 1.76 NA P9 9.11, 8.55, 6.94, 6.91, 4.95, 4.18, 4.11, 3.84, 3.37, 3.02, 3.01, 2.93, 2.62, 2.24, 2.19, 2.00, 1.63, 1.52, 1.32, 1.23 NA P10 9.10, 8.62, 7.23, 7.15, 6.91, 4.29, 4.17, 4.14, 3.34, 3.23, 3.22, 3.20, 3.15, 3.14, 2.68, 2.40, 2.26, 1.97, 1.67, 1.33 NA

DMD # 33670T



this version.D

MD



I: 10.1124/dmd.110.033670



32

Table 5. Concentrations (ng equivalent/mL) and percentage of total radioactivity (in parentheses) of vabicaserin and metabolites in plasma of male mice, rats and dogs following a single oral administration.

Species Mice Rats Dogs Time (h) 2 4 8 2 4 8 4 8 24

Vabicaserin 0.58 (15.1) 0.46 (12.3) 0.38 (11.7) 86.9 (13.7) 90.8 (13.8) 58.7 (12.7) 90.2 (16.0) 4.75 (0.9) 21.9 (1.6) P1 ND ND ND 88.4 (14.0) 59.7 (9.1) 41.9 (8.9) 36.4 (6.5) 39.4 (7.5) 68.3 (5.1) P2 0.51 (13.4) 0.55 (14.7) 0.41 (12.7) 32.3 (5.2) 22.6 (3.4) 20.0 (4.3) 52.5 (9.3) ND 53.6 (4.0)

P3/M9 0.53 (13.8) 0.57 (15.0) 0.65 (19.8) 76.4 (12.1) 105 (16.0) 64.2 (13.8) 135 (24.0) 364 (69.0) 875 (65.3) P4 ND ND ND 28.3 (4.4) 28.3 (4.3) 12.4 (2.5) ND ND ND P5 ND ND ND ND ND ND 33.6 (6.0) 7.04 (1.3) ND P7 0.25 (6.6) 0.25 (6.5) 0.19 (5.7) 63.6 (10.1) 62.1 (9.4) 53.5 (11.5) 41.5 (7.4) ND 88.0 (6.6) P8 ND ND ND 15.1 (2.4) 30.3 (4.6) ND ND ND ND

P10 ND ND ND 11.6 (1.9) 14.2 (2.2) 6.33 (1.4) ND ND ND P11 ND ND ND 23.7 (3.6) 9.81 (1.5) 13.2 (2.9) ND ND ND P12 ND ND ND 37.2 (6.0) 34.5 (5.2) 16.3 (3.6) ND ND ND P13 ND ND ND ND ND ND 51.0 (9.1) 27.5 (5.2) ND P14 0.3 (7.9) 0.31 (8.2) 0.26 (8.0) ND ND ND CG 0.57 (14.9) 0.78 (20.6) 0.51 (15.7) ND ND ND 36.7 (6.5) 12.3 (2.3) 101 (7.5)

Others a 3.82 (28.3) 3.77 (22.7) 3.26 (26.5) 168 (26.4) 201 (31.0) 179 (38.4) 88.0 (15.6) 72.0 (13.6) 133 (9.9) Concentrations were estimated based on the total plasma radioactivity concentrations and the percent distribution of plasma radioactivity. a, sum of a number of uncharacterized minor metabolites each representing less than 5% of plasma radioactivity; ND, not detected.

DMD # 33670T



this version.D

MD



I: 10.1124/dmd.110.033670



33

Table 6. Distribution (% of dose) of vabicaserin and metabolites in urine of mice, rats and dogs.a Species vabicaserin P1 P2 b P3/P9 P4 P5 P7 P8 P11 P13 P14 P15 P16 P17 P18 CG Othersc Total Mouse 1.2 ND 19.2 7.1 ND ND 4.8 ND ND ND 3.2 1.9 3.0 2.3 ND 17.0 59.7

Rat 0.9 16.4 5.1 12.6 2.2 ND 6.6 2.6 2.0 ND ND ND ND ND ND ND 19.0 67.5 Dog 0.5 1.4 5.9 3.5 ND 0.3 0.8 ND ND 1.1 ND ND ND 2.1 0.9 1.9 8.7 26.9

a, Data represent 0-24 h for mice and rats and 0-48 h for dogs following oral administration; b, P19 co-eluted with P2 in mouse urine; c, A sum of a number of uncharacterized minor metabolites each representing less than approximately 3% of dose for mouse and rat, and 1.5% of dose for dog.

DMD # 33670T



this version.D

MD



I: 10.1124/dmd.110.033670




at ASPE

T Journals on A

ugust 4, 2018dm


ownloaded from



at ASPE

T Journals on A

ugust 4, 2018dm


ownloaded from



at ASPE

T Journals on A

ugust 4, 2018dm


ownloaded from



at ASPE

T Journals on A

ugust 4, 2018dm


ownloaded from



at ASPE

T Journals on A

ugust 4, 2018dm


ownloaded from



at ASPE

T Journals on A

ugust 4, 2018dm


ownloaded from



at ASPE

T Journals on A

ugust 4, 2018dm


ownloaded from



at ASPE

T Journals on A

ugust 4, 2018dm


ownloaded from



at ASPE

T Journals on A

ugust 4, 2018dm


ownloaded from


Date post:	04-Aug-2018
Category:	Documents
Upload:	voxuyen
View:	219 times
Download:	0 times

METABOLISM OF VABICASERIN IN MICE, RATS,...

Documents