Disposition and Metabolic Profiling of [14C] Cerlapirdine Utilizing

DMD Fast Forward. Published on September 12, 2014 as DOI: 10.1124/dmd.114.059675 This article has not been copyedited and formatted. The final version may differ from this version.

DMD #59675

Disposition and Metabolic Profiling of [14C]Cerlapirdine Utilizing Accelerator Mass Spectrometry (AMS)

Susanna Tse, Louis Leung, Sangeeta Raje, Mark Seymour, Yoko Shishikura, R. Scott

Obach

Pfizer Inc., Groton, CT (S.T., L.L., R.S.O.) and Collegeville PA (S.R.); Xceleron Inc.,

Germantown, MD (M.S., Y.S.) Downloaded from

dmd.aspetjournals.org at ASPET Journals on September 26, 2021

DMD Fast Forward. Published on September 12, 2014 as DOI: 10.1124/dmd.114.059675 This article has not been copyedited and formatted. The final version may differ from this version.

DMD #59675

Running Title: Metabolism of Cerlapirdine in Humans

Address Correspondence to:

Susanna Tse

Pharmacokinetics, Dynamics and Metabolism-New Chemical Entities

Pfizer Inc.

Eastern Point Road Downloaded from

Groton, CT 06340, USA.

Telephone number: 1-860-715-5942 dmd.aspetjournals.org Fax number: 1-860-715-6427

Email: [email protected]

at ASPET Journals on September 26, 2021

Text Pages (including references): 36

Tables: 3

Figures: 9

References: 44

Words in:

Abstract: 242

Introduction: 617

Discussion: 1198

DMD Fast Forward. Published on September 12, 2014 as DOI: 10.1124/dmd.114.059675 This article has not been copyedited and formatted. The final version may differ from this version.

DMD #59675

Abbreviations used: PF-05212365: N,N-dimethyl-3-{[3-(naphthalen-1-ylsulfonyl)-2H- indazol-5-yl]oxy}propan-1-amine; M1: N-desmethylcerlapirdine, N-methyl-3-{[3-

(naphthalen-1-ylsulfonyl)-2H-indazol-5-yl]oxy}propan-1-amine; M3: cerlapirdine-N- oxide; 5-HT: 5-hydroxytryptamine; ADME: absorption, distribution, metabolism and excretion; GMP: Good Manufacturing Practices; AMS: accelerator mass spectrometry;

HPLC: high performance liquid chromatography; LC/MS-MS: liquid chromatography tandem mass spectrometry; m/z: mass to charge ratio; CYP: cytochrome P450; rCYP: Downloaded from recombinant cytochrome P450; CLint: intrinsic clearance; HLM: human liver microsomes; PK: pharmacokinetics; Cmax: peak plasma concentration; tmax: time at peak dmd.aspetjournals.org plasma concentration; AUC: area under the concentration-time curve; λz: terminal-phase disposition rate constant; t½: apparent terminal half-life; CL/F: apparent oral clearance;

Vz/F: apparent volume of distribution at ASPET Journals on September 26, 2021

DMD Fast Forward. Published on September 12, 2014 as DOI: 10.1124/dmd.114.059675 This article has not been copyedited and formatted. The final version may differ from this version.

DMD #59675

ABSTRACT

Cerlapirdine (SAM-531, PF-05212365) is a selective, potent, full antagonist of the 5- hydroxytryptamine 6 (5-HT6) receptor. Cerlapirdine and other 5-HT6 receptor antagonists have been in clinical development for the symptomatic treatment of Alzheimer’s Disease.

A human ADME study was conducted to gain further understanding of the metabolism and disposition of cerlapirdine. Due to the low amount of radioactivity administered, total

14C content and metabolic profiles in plasma, urine and feces were determined using Downloaded from accelerator mass spectrometry (AMS). After a single, oral 5 mg dose of [14C]cerlapirdine

(177 nCi), recovery of total 14C was almost complete, with feces being the major route of dmd.aspetjournals.org elimination of the administered dose, whereas urinary excretion played a lesser role. The extent of absorption was estimated to be at least 70%. Metabolite profiling in pooled plasma samples showed that unchanged cerlapirdine was the major drug-related at ASPET Journals on September 26, 2021 component in circulation, representing 51% of total 14C exposure in plasma. One (1) metabolite (M1, desmethylcerlapirdine) was detected in plasma, and represented 9% of total 14C exposure. In vitro cytochrome P450 reaction phenotyping studies showed that

M1 was formed primarily by CYP2C8 and CYP3A4. In pooled urine samples, three major drug-related peaks were detected, corresponding to cerlapirdine-N-oxide (M3), cerlapirdine, and desmethylcerlapirdine. In feces, cerlapirdine was the major 14C component excreted, followed by desmethylcerlapirdine. The results of this study demonstrate that the use of the AMS technique enables comprehensive quantitative elucidation of the disposition and metabolic profiles of compounds administered at a low radioactive dose.

DMD Fast Forward. Published on September 12, 2014 as DOI: 10.1124/dmd.114.059675 This article has not been copyedited and formatted. The final version may differ from this version.

DMD #59675

INTRODUCTION

Serotonin (5-hydroxytryptamine, 5-HT) is a major neurotransmitter in both the central and peripheral nervous systems. At least seven 5-HT receptor families (5-HT1-7) have been identified (Hoyer et al., 1994). Among the 5-HT receptor families, the 5-HT6 receptor is localized primarily in the central nervous system (CNS), and in regions including the cerebral cortex, nucleus accumbens, caudate-putamen, striatum and hippocampus (Roberts et al., 2002; Hirst et al., 2003; Marazziti et al., 2012), which are Downloaded from associated with cognition and memory (Nyberg et al., 1996; Reinvarg et al., 1998;

Burgess et al., 2001). In experimental models, 5-HT6 antagonists have demonstrated dmd.aspetjournals.org ability to increase brain levels of acetylcholine and glutamate (Dawson et al., 2000;

Riemer et al., 2003; Marcos et al., 2006), and improve cognitive functions in animal models (Lindner et al., 2003; Foley et al., 2004; Lieben et al., 2005; Arnt et al., 2010; at ASPET Journals on September 26, 2021

Mohler et al., 2012). Several 5-HT6 antagonists have been evaluated in clinical trials for the treatment of cognitive deficits and dementia associated with Alzheimer’s Disease

(Upton et al., 2008; Johnson et al., 2008; Geldenhuys and Van der Schyf, 2009). 5-HT6 antagonists have also been proposed to have utility as an adjunctive treatment of cognitive dysfunction in schizophrenia (Roth et al., 2004; Schreiber et al., 2006) and other neurological diseases (Liu and Robichaud, 2009; Rosse and Schaffhauser, 2010;

Codony et al., 2011). Recent clinical evidence further illustrates the potential utility of 5-

HT6 antagonists in the symptomatic treatment of Alzheimer’s Disease (Maher-Edwards et al., 2010, 2011). Cerlapirdine (SAM-531, PF-05212365) is a selective, potent, full antagonist at the 5-HT6 receptor (Liu and Robichaud, 2010) with pro-cognitive effects in nonclinical species (Comery et al., 2010). Clinical safety, tolerability and preliminary

DMD Fast Forward. Published on September 12, 2014 as DOI: 10.1124/dmd.114.059675 This article has not been copyedited and formatted. The final version may differ from this version.

DMD #59675 efficacy studies have been conducted (Baird-Bellaire et al., 2010; Brisard et al., 2010;

Mitchell, 2011). A study using radiolabeled [14C]cerlapirdine was conducted in healthy young male subjects to gain a comprehensive understanding of the absorption, metabolism and excretion properties of cerlapirdine. Furthermore, in vitro metabolism and reaction phenotyping studies were conducted to elucidate the metabolic pathways and the oxidative enzymes responsible for the formation of the major metabolite that was observed in vivo. Downloaded from

Accelerator mass spectrometry (AMS) is an extremely sensitive method for the detection dmd.aspetjournals.org and determination of isotopic ratios (Salehpour et al, 2008). AMS has been utilized in pharmaceutical research and development since 2000 (Garner, 2000), including applications in pharmacokinetic (Sarapa et al, 2005; Hah, 2009) and metabolism studies at ASPET Journals on September 26, 2021

(Lappin and Stevens, 2008). In some radiotracer studies in which very low amounts of radioactivity may be administered due to various reasons, such as radiolytic instability, long plasma half-lives or retention in tissues, the ultra-high sensitivity of AMS enables the detection of very low levels of radio-isotopes which may not be detectable by conventional techniques. In the case of cerlapirdine, prior to the conduct of the ADME study in humans using [14C]cerlapirdine, dosimetry calculations were performed to evaluate the risk involved with exposure to ionizing radiation in human subjects, based on in vivo distribution, PK and mass balance studies in rats (data on file). Based on the absorption and distribution of radioactivity to the whole body and major tissues, which indicated prolonged retention of radioactivity in pigmented tissues, a single, low oral radioactive dose of ~200 (actual dose 177) nCi of [14C]cerlapirdine was selected for

DMD Fast Forward. Published on September 12, 2014 as DOI: 10.1124/dmd.114.059675 This article has not been copyedited and formatted. The final version may differ from this version.

DMD #59675 administration in the human study. Due to the low amount of radioactivity anticipated in plasma and in excreta, which were likely to be below the levels that can be reliably measured by liquid scintillation counting, levels of total 14C in plasma, whole blood, urine and feces were determined using AMS. Subsequently, metabolic profiles of

[14C]cerlapirdine in plasma, urine and feces were determined in pooled samples, using

HPLC fractionation followed by AMS analysis of fractions (HPLC+AMS).

Downloaded from

MATERIALS & METHODS

Materials dmd.aspetjournals.org [14C]cerlapirdine (Figure 1) was prepared under GMP conditions by ABC Labs at

Columbia, MO. Cerlapirdine and [14C]cerlapirdine were blended and co-crystallized prior to the preparation of the final (capsule) dosage form. Each capsule was hand-filled, at ASPET Journals on September 26, 2021 weighed and capped. Each dose was packed in one capsule per subject, with a target dose of 5.44 mg of the hydrochloride salt or approximately 4.96 mg of active moiety (free base). Each dose vial contained approximately 200 nCi (~7.4 kBq; actual mean radioactive dose was determined to be 177 nCi) of [14C]cerlapirdine. Authentic standards of cerlapirdine, desmethylcerlapirdine (M1, N-desmethyl metabolite), N-oxide metabolite

2 (M3) of cerlapirdine, [ H6] cerlapirdine (internal standard for cerlapirdine in LC/MS-MS

2 method), [ H6]desmethylcerlapirdine (internal standard for desmethylcerlapirdine in

LC/MS-MS method) and WAY-211560-5 (internal standard used for reaction phenotyping study) were obtained from Pfizer Global Research & Development, Pearl

River, NY or Princeton, NJ.

DMD Fast Forward. Published on September 12, 2014 as DOI: 10.1124/dmd.114.059675 This article has not been copyedited and formatted. The final version may differ from this version.

DMD #59675

The following materials were used for the LC/MS-MS analyses of plasma, urine, fecal samples and metabolite profiling and identification: methanol, acetonitrile, methyl-t-butyl ether and ethyl acetate were obtained from Burdick & Jackson; isopropyl alcohol and sodium borate 10-hydrate were obtained from J.T. Baker; formic acid Superpur® was obtained from EMD Chemicals. Materials used for the graphitization process, AMS and

HPLC+AMS analysis: methanol (HPLC and AR grades) was obtained from Fisher

Scientific; liquid paraffin, copper oxide wire (ACS grade), cobalt powder (100 Mesh, Downloaded from

99.9%), zinc powder (100 Mesh, 99%), titanium (II) hydride (325 Mesh, 98%) were obtained from Sigma-Aldrich Chemical Co.; ANU sugar (certified 14C:12C ratio = 1.5061 dmd.aspetjournals.org Times Modern) used as an AMS standard was obtained from Quaternary Dating Research

Centre, Australian National University, Canberra, Australia; synthetic graphite 200-35

Mesh (99.99%) was obtained from Alfa Aesar (Johnson Mathey PLC) and contained at ASPET Journals on September 26, 2021 aluminium powder (99.99%, obtained from Acros Organics); solid aluminium cathode

(used as machine control) was obtained from National Electostatics Corp., USA; graphitization tubes, samples tubes and borosilicate glass tubes (pre-baked at 500ºC for 2 to 4 h) were obtained from York Glassware Services Ltd. UK; tin capsules and

Chromosorb W were obtained from Elemental Microanalysis Ltd., UK; urea standard was obtained from ThermoQuest.

The following materials were used for in vitro cytochrome P450 reaction phenotyping experiments: sulfaphenazole, tranylcypromine, quinidine, ketoconazole, quercetin,

α-naphthoflavone and diethyldithiocarbamate were purchased from Sigma Chemical Co.

(St. Louis, MO). HPLC grade water, methanol, and acetonitrile were obtained from

DMD Fast Forward. Published on September 12, 2014 as DOI: 10.1124/dmd.114.059675 This article has not been copyedited and formatted. The final version may differ from this version.

DMD #59675

E.M. Science (Gibbstown, NJ). All other chemicals were reagent grade or better. Human liver microsomes (pool of 50 male donors) were purchased from XenoTech LLC

(Kansas City, KS). Recombinant human CYP enzymes CYP1A2, CYP2A6, CYP2C8,

CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4 expressed in E. coli were prepared at Pfizer Inc., Collegeville, PA.

Downloaded from Dosing of Subjects

This was an open-label, non-randomized, single dose study conducted at a single clinical

site (PRA International EDS, The Netherlands), in accordance with the International dmd.aspetjournals.org

Conference on Harmonisation (ICH) guideline of Good Clinical Practice (GCP), and ethical principles as per the Declaration of Helsinki. The clinical protocol and Informed

Consent Form (ICF) were reviewed and approved by the Institutional Review Board at ASPET Journals on September 26, 2021

(IRB) and Independent Ethics Committee (IEC) prior to subject enrollment. Subjects were fasted for 10 hours prior to dose administration, and fasted for 4 hours after dosing.

On study day 1, each of six (6) healthy Caucasian male volunteers (age 19-48, mean age

27; weight 66.3-89.0 kg, mean weight 76.8 kg) was administered a single capsule containing a 5 mg (free base) oral dose of [14C] cerlapirdine (177 nCi radioactive dose) at approximately 8:00 AM, and received 240 ml of room temperature water immediately after dosing. All six study subjects were discharged on day 15 after the 14-day sample collection and study period.

Collection of Samples

DMD Fast Forward. Published on September 12, 2014 as DOI: 10.1124/dmd.114.059675 This article has not been copyedited and formatted. The final version may differ from this version.

DMD #59675

Blood (venous) samples were collected prior to drug administration (pre-dose sample) and at 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 16, 24, 48, 72, 96, 120, 144, 168, 192, 216, 240, 264,

288, 312 and 336 hours after drug administration. Potassium EDTA was used as an anticoagulant. Each sample was gently mixed and stored on ice until centrifugation.

Aliquots of each blood sample were centrifuged at 2500 RPM for 10 minutes at 4ºC to obtain plasma. The plasma was removed and stored frozen at -70ºC/-80ºC in polypropylene tubes prior to and after analysis. Downloaded from

Urine samples were collected prior to drug administration (pre-dose specimen) and at 0- dmd.aspetjournals.org 4, 4-8, 8-12, 12-24, 24-48, 48-72, 72-96, 96-120, 120-144, 144-168, 168-192, 192-216,

216-240, 240-264, 264-288, 288-312, 312-336 hours after dosing. Urine samples were collected on wet ice during each collection period, then frozen and stored at -70ºC/-80ºC at ASPET Journals on September 26, 2021 prior to and after analysis. Fecal samples were collected prior to drug administration (pre- dose specimen) and then all bowel movements were collected following drug administration up to 336 hours after dosing. Fecal samples were quantitatively transferred into a container per subject per 24 hour interval (pre-dose, 0-24, 24-48 h etc.) and the total weight for each sample at each interval was recorded. The fecal samples were homogenized with an Ultra Turax mixer after adding one to two weight equivalents of water. Aliquots of fecal homogenates were analyzed for metabolite profiling

(HPLC+AMS) and metabolite identification (LC/MS-MS). Separate sub-samples of fecal homogenates were freeze-dried and analyzed for 14C content (AMS) in the mass balance determination. The total amount of feces collected and the time of each defecation relative to the time of drug administration were recorded. Fecal homogenate

DMD Fast Forward. Published on September 12, 2014 as DOI: 10.1124/dmd.114.059675 This article has not been copyedited and formatted. The final version may differ from this version.

DMD #59675 and freeze-dried feces samples were stored in plastic containers at -70ºC/-80ºC prior to and after analysis.

Accelerator Mass Spectrometry (AMS) Analyses

Whole blood, plasma, urine and fecal samples were assayed for total 14C content using

AMS at Xceleron Inc., York, UK. Metabolite profiling of pooled plasma, urine and fecal samples were conducted using HPLC with offline 14C detection by AMS at Xceleron Downloaded from

Inc., York, UK. For the AMS analysis, plasma (60 µl), whole blood (20 µl), urine

(diluted 10-fold with water; 100 µl), feces (homogenized, ca 40 mg; freeze-dried, ca 4 dmd.aspetjournals.org mg), HPLC fractions (100 µl) or fraction pools (250 µl) were placed in tubes containing

CuO2. For samples containing little carbon (i.e. urine and HPLC fractions), liquid paraffin (equivalent to ca 2 mg carbon) was added as carbon carrier. Samples were sealed at ASPET Journals on September 26, 2021 into quartz tubes, under vacuum, heated for 2 h at 900°C, oxidizing all carbon in the sample to CO2. CO2 was cryogenically transferred and sealed into a glass tube containing

TiH2 and Zn, with cobalt powder as catalyst. Samples were heated for 4 h at 500°C, then for 6 h at 550°C, reducing CO2 to solid carbon. Carbon was pressed into aluminum cathodes, which were placed in the ion source of a 5MV tandem pelletron Accelerator

Mass Spectrometer (Model 15SDH-2; National Electrostatics Corp, Middleton, WI), ionized using a Cs+ ion beam and the 14C:12C and 13C:12C ratios were determined. The 14C content of each sample was calculated, based on the 14C:12C ratio and the total carbon content. Carbon content was measured using an NA2100 Brewanalyser (CE Instruments,

Wigan, UK) or generic values were used. Where appropriate, pre-dose samples from the same subject were graphitized and analyzed to determine background 14C:12C ratios.

DMD Fast Forward. Published on September 12, 2014 as DOI: 10.1124/dmd.114.059675 This article has not been copyedited and formatted. The final version may differ from this version.

DMD #59675

Mass Balance and Blood:Erythrocyte Distribution

Mass balance was determined based on the recovery of total 14C in urine and feces.

Distribution of blood radioactivity to erythrocytes at selected time points (up to 96 h post-

14 dose) was calculated from C contents in blood (Cblood), plasma (Cplasma) and hematocrit

(Hct) using the formula: (Cblood-Cplasma)(1-Hct)/Hct. Ratios of plasma cerlapirdine and M1

AUC to total plasma 14C AUC were also determined as follows: Downloaded from

14 Cerlapirdine or M1 AUC ratio = AUCcerlapirdine or AUCM1/AUC[total C]

dmd.aspetjournals.org LC/MS-MS Analyses of Cerlapirdine and Desmethylcerlapirdine (M1) in Human Plasma

The concentrations of cerlapirdine and desmethylcerlapirdine (M1) in plasma and urine samples were determined at Advion BioServices, Inc. (Ithaca, NY) using validated at ASPET Journals on September 26, 2021

LC/MS-MS assays. Plasma or urine samples (100 µl) were extracted by a liquid-liquid extraction procedure using a 96-well format to isolate cerlapirdine and

2 2 desmethylcerlapirdine. [ H6]cerlapirdine and [ H6]desmethylcerlapirdine were used as internal standards (IS) for cerlapirdine and desmethylcerlapirdine, respectively. Sample extracts were injected on a 2.0 x 50 mm, 80 Å, (4 µm particle size) Synergi Hydro-RP column (Phenomenex Inc., Torrance, CA). A mobile phase gradient starting at 80% mobile phase A (10:90:0.01 methanol:water:formic acid) and 20% mobile phase B

(90:10:0.01 methanol:water:formic acid) was used. Cerlapirdine and desmethylcerlapirdine concentrations in plasma extracts were determined by a Sciex API

4000 mass spectrometer with turbo ion spray in the positive ionization mode. The plasma method has a lower limit of quantitation (LLOQ) of 0.2 ng/ml and a calibration curve

DMD Fast Forward. Published on September 12, 2014 as DOI: 10.1124/dmd.114.059675 This article has not been copyedited and formatted. The final version may differ from this version.

DMD #59675 range of 0.2 to 100 ng/ml for both analytes. cerlapirdine and desmethylcerlapirdine concentrations in urinary extracts were determined by a Sciex API 3000 mass spectrometer (ABSciex, Framingham, MA) with turbo ion spray in the positive ionization mode. The urinary method has a lower limit of quantitation (LLOQ) of 10 ng/ml and a calibration curve range of 10 to 500 ng/ml.

In Vitro Cytochrome P450 Reaction Phenotyping Downloaded from

Experiments were conducted to determine the intrinsic clearance by substrate depletion of cerlapirdine or formation of desmethylcerlapirdine in human liver microsomes. dmd.aspetjournals.org Incubations were conducted at 37°C with shaking on Thermomixer R (Eppendorf) in

96-well square plates containing (1 μM) in potassium phosphate buffer (100 mM, pH

7.4), MgCl2 (10 mM), human liver microsomes (1 mg/ml) and were initiated with the at ASPET Journals on September 26, 2021 addition of a NADPH regenerating system (glucose-6-phosphate, 3.6 mM; NADP+,

1.3 mM and glucose-6-phosphate dehydrogenase, 0.4 units/ml) in the absence and presence of selective inhibitors of cytochrome P450 enzymes including ketoconazole (1 μM) for CYP3A, quinidine (10 μM) for CYP2D6, sulfaphenazole (10 μM) for CYP2C9, tranylcypromine (50 μM) for CYP2C19,

α-naphthoflavone (5 μM) for CYP1A2, tranylcypromine (10 μM) for CYP2A6, quercetin (20 μM) for CYP2C8 or diethyldithiocarbamate (50 μM) for CYP2E1.

Reactions were quenched by the addition of 0.5 ml acetonitrile containing the internal standard (WAY-211560). The plates were then centrifuged in a Thermo Forma

(Marietta, OH) centrifuge at room temperature for 10 min at 3400 rpm. Supernatants

(200 µl) were transferred to 1 ml polypropylene 96-well plates for the determination of

DMD Fast Forward. Published on September 12, 2014 as DOI: 10.1124/dmd.114.059675 This article has not been copyedited and formatted. The final version may differ from this version.

DMD #59675 the parent drug or M1 concentrations by LC/MS-MS. Similar incubations were conducted in recombinant human cytochrome P450 enzymes CYP1A2, CYP2A6, CYP2C8,

CYP2C9, CYP2C19, CYP2D6, CYP2E1 or CYP3A4 (100 pmol/ml) expressed in E. coli.

The kinetics of desmethylcerlapirdine formation were determined in recombinant

CYP2C8 and CYP3A4 based on results from an initial study, and in human liver microsomes at parent drug concentrations of 1, 5, 10, 25 and 50 μM similarly for an incubation duration of 10 minutes. Downloaded from

HPLC analysis was carried out on an Agilent model 1100 HPLC equipped with a binary dmd.aspetjournals.org pump (Agilent Technologies, Santa Clara, CA) and using an HTS PAL autosampler

(LEAP Technologies, Raleigh, NC) maintained at 10°C. Aliquots (10 μl) of the acetonitrile supernatants (containing cerlapirdine, desmethylcerlapirdine and the internal at ASPET Journals on September 26, 2021 standard) were injected onto the HPLC. The column was a 5 μm Thermo Aquasil C18 column, 2.1 mm ID × 50 mm (Bellefonte, PA). Mobile phase A was 0.1% formic acid in water and mobile phase B was 0.1% formic acid in acetonitrile. Mass spectrometry analyses were conducted using an Applied Biosystems PE Sciex API 4000 triple quadrupole mass spectrometer (Applied Biosystems, Foster City, CA), with a turbo spray interface using the positive electrospray ionization (ESI) mode with ESI source utilizing a multiple reaction monitoring (MRM) technique. Data analysis was performed using

Analyst™, ver. 1.4.1. Peak area ratios for the parent drug against the internal standard were used to express the amount of parent remaining compared to that at time zero in both human liver microsomes with or without chemical inhibitors and in recombinant

CYP enzymes. Intrinsic clearance by substrate depletion was determined by linear

DMD Fast Forward. Published on September 12, 2014 as DOI: 10.1124/dmd.114.059675 This article has not been copyedited and formatted. The final version may differ from this version.

DMD #59675 regression of the log % substrate remaining versus time plots using Microsoft Excel, ver.

7.0. Concentrations of desmethylcerlapirdine formed were determined by extrapolating the response calculated from the peak-area ratios (peak areas of desmethylcerlapirdine versus the internal standard) to that of the standard curve. Vmax and Km values were determined by non-linear regression in plots generated from the simple Emax model

(model 101) utilizing WinNonlin Professional, version 4.1 (Pharsight, Mountain View,

CA). The intrinsic clearance (Vmax /Km) of desmethylcerlapirdine formation determined Downloaded from in a recombinant human CYP (rCYP) enzyme was converted to the intrinsic clearance in human liver microsomes using the relative activity factor (RAF). The RAF is the ratio of dmd.aspetjournals.org the specific activity in human liver microsomes to the activity by the recombinant CYP enzyme (Nakajima et al., 2002). The specific activity is measured as intrinsic clearance

(Vmax/Km) using a specific probe substrate in human liver microsomes and in the at ASPET Journals on September 26, 2021 recombinant CYP enzyme for the CYP enzyme of interest:

RAF = CL int HLM / CL int rCYP

or CL int HLM = RAF x CL int rCYP

The RAF values for CYP2C8 and CYP3A4 determined internally were 0.327 and

0.0432 nmol CYP/mg protein, respectively. The % contribution of the intrinsic clearance by a particular CYP enzyme (CL int, CYP i HLM) to total CYP-mediated intrinsic clearance

(CL int, CYP sum HLM) in human liver may then be calculated as follows:

% CYPi Contribution = 100% × CL int, CYP i HLM / CL int, CYP sum HLM

Metabolite Profiling in Plasma, Urine and Feces

DMD Fast Forward. Published on September 12, 2014 as DOI: 10.1124/dmd.114.059675 This article has not been copyedited and formatted. The final version may differ from this version.

DMD #59675

Plasma, urine and fecal homogenate samples were pooled across all six subjects at selected time points. A single 0-72h plasma pooled sample was prepared from aliquots of plasma samples at each time point according to the Hamilton method (Hamilton et al.,

1981). Two pooled urine samples (0-96h and 96-168h) were prepared by combining volumes of individual time point samples proportional to the volume collected over each time period for each subject. Two pooled feces samples (0-96h and 96-168h) were prepared for each subject by combining a mass of each individual sample proportional to Downloaded from the total mass excreted over each time period by that subject. An equal proportion of each individual subject feces pool was then pooled, to provide two cross-subject pooled dmd.aspetjournals.org samples (0-96h and 96-168h) for analysis.

Plasma extracts for metabolite profiling were prepared by adding 1 ml of pooled plasma at ASPET Journals on September 26, 2021 sample to 3 ml of acetonitrile, vortex-mixing then centrifuging (4500 rpm; 10 min; ambient temperature). The supernatant was transferred and evaporated under a stream of nitrogen to approximately 0.6 ml, followed by addition of 0.4 ml of 5% v/v acetonitrile in

0.2% v/v formic acid. Aliquots of pooled urine samples were centrifuged and metabolite profiles were determined in the supernatants. Fecal homogenate samples for metabolite profiling were prepared by mixing approximately 200 mg of each overall pooled homogenate sample with 1 ml of water. Acetonitrile (4 ml) was added to 0.8 ml of the mixture, vortex-mixed and centrifuged. The supernatant was transferred to a clean tube and evaporated under a stream of nitrogen to approximately 0.6 ml, followed by adding

0.4 ml of 5% v/v acetonitrile in 0.2% v/v formic acid. Plasma, urine and feces metabolite profiling samples were spiked with reference standards (cerlapirdine;

DMD Fast Forward. Published on September 12, 2014 as DOI: 10.1124/dmd.114.059675 This article has not been copyedited and formatted. The final version may differ from this version.

DMD #59675 desmethylcerlapirdine, M1; and cerlapirdine-N-oxide, M3) then fractionated by HPLC.

Metabolite peaks were separated on an YMC ODS-AQ column (4.6 x 250 mm, 5 µm) at a temperature set at 30ºC. Mobile phase A consisted of 0.2% v/v formic acid in water, and mobile phase B consisted of 0.2% v/v formic acid in acetonitrile. The flow rate was 1 ml/min and peaks were separated using a stepwise gradient from 15% to 95% mobile phase B over 58 minutes (15% to 20%B from 0 to 20 min, held at 20%B for 10 min, linear gradient to 30%B over 20 min, then to 95%B over 5 min and held at 95%B for 3 Downloaded from min before column re-equilibration at 15%B). The eluent was collected as a series of fractions at 15 second intervals across each run. Fractions were analyzed either dmd.aspetjournals.org individually or pooled then analyzed for 14C content by AMS as described above.

Metabolite Identification at ASPET Journals on September 26, 2021

Metabolite peaks from plasma, urinary and fecal profiling samples were collected using a fraction collector, and were analyzed by positive ion HPLC-UV-MS using a Thermo

Finnigan LTQ-Orbitrap (Thermo Quest Inc.) in a full scan mode (m/z 200-650) at a resolution setting of 30000. Capillary temperature was set at 275°C and the source potential was 3600V. Other potentials were optimized to get optimal ionization and fragmentation of the parent compound. UV absorption spectra were obtained by an in- line Surveyor photodiode array detector. A Polaris (Varian) C18 column was used (4.6 x

250 mm) with a flow rate of 0.8 ml/min. Mobile phase A was comprised of 0.1% formic acid, and mobile phase B was comprised of acetonitrile. The gradient system used was as follows: initially, 10% B held for 5 minutes followed by a linear gradient to 50% B at 50 minutes, washing the column at 95% B for 10 minutes, followed by column re-

DMD Fast Forward. Published on September 12, 2014 as DOI: 10.1124/dmd.114.059675 This article has not been copyedited and formatted. The final version may differ from this version.

DMD #59675 equilibration to initial conditions for 5 min. Collision induced dissociation spectra were obtained for m/z 410, 396, and 426. Proposed structures were compared to available authentic standards of cerlapirdine, desmethylcerlapirdine and cerlapirdine-N-oxide.

Parent and Metabolite Excretion in Urine and Feces

Amounts of cerlapirdine and observed metabolites excreted in urine and feces were calculated by multiplying urine volume or feces weight and cerlapirdine (or metabolites) Downloaded from concentration in pooled urine or fecal homogenate (adjusted by homogenate volume) sample at each time interval. The total urinary or fecal excretion of cerlapirdine (or dmd.aspetjournals.org metabolites) was obtained by summation across all time intervals. Percent dose urinary or fecal excretion of cerlapirdine (or metabolites) was calculated by dividing total amount excreted in urine or feces by the administered dose and multiplying by 100. at ASPET Journals on September 26, 2021

Pharmacokinetic and Statistical Analyses

The pharmacokinetics of total 14C, cerlapirdine and desmethylcerlapirdine in plasma were analyzed using WinNonlin (version 5.1.1, Pharsight, Mountain View, CA). PK parameters were calculated by noncompartmental analysis using the linear-up/log-down trapezoidal method. Based on the 336-hour plasma total 14C, cerlapirdine and desmethylcerlapirdine concentration data, peak plasma concentration (Cmax) and time at peak plasma concentration (tmax) values were taken directly from the plasma concentration vs. time curve for each subject. Terminal-phase disposition rate constant

(λz) was determined by the log-linear regression of at least 3 time points (maximum of 5 time points) that were judged to be in the terminal phase. The apparent terminal half-life

DMD Fast Forward. Published on September 12, 2014 as DOI: 10.1124/dmd.114.059675 This article has not been copyedited and formatted. The final version may differ from this version.

DMD #59675

(t½) value was estimated by dividing 0.693 by λz. Total area under the concentration-time curve to the last measureable concentration at time t (AUCt) was determined by the log- linear trapezoidal rule from time 0 to the time of last observed concentration at time t

(Ct). Total area under the concentration-time curve from time zero to infinity (AUCinf) was determined by: AUCinf=AUCt+Ct/λz. CL/F was calculated as dose/total area under the concentration-time curve (AUC), and Vz/F was determined by the following: Vz/F =

(dose/AUC) x (1/ λz). CL/F and Vz/F were calculated only for the parent compound Downloaded from cerlapirdine.

dmd.aspetjournals.org Statistical analysis was limited to calculation of summary statistics (e.g. mean, standard deviation [SD] and range) of the PK parameters across the study subjects. Since this study was conducted in a single study group with a single treatment, no statistical at ASPET Journals on September 26, 2021 comparison was performed.

RESULTS

Excretion and Mass Balance

The study population consisted of 6 (six) healthy, male, Caucasian subjects aged 19 to 48 years, with a mean age of 27 years. A summary of the subject demographic and baseline characteristics are summarized in Table 1. The excretion of 14C in urine, feces and total excretion with time is shown in Figure 2. After a single oral dose of [14C]cerlapirdine, excretion was essentially complete, with mean (±SD) total 14C recovery of 98.3 (±4.7)%

(range: 91.0% to 103%) up to 336 hours post-dose. Feces was the major route of excretion, accounting for 70.3 (±5.1)% (range: 63.8% to 76.9%) of the administered dose,

DMD Fast Forward. Published on September 12, 2014 as DOI: 10.1124/dmd.114.059675 This article has not been copyedited and formatted. The final version may differ from this version.

DMD #59675 and urine was the minor excretory route, accounting for 28.0 (±5.2)% (range: 24.4% to

37.8%) of the administered dose. The rate of excretion was rapid, with the majority

(~68%) of the 14C dose recovered within 96 hours.

Pharmacokinetic Profiles of total 14C, Cerlapirdine and Desmethylcerlapirdine (M1)

The plasma and blood concentrations vs. time profiles of total 14C are presented in Figure

3. Pharmacokinetic parameters of total 14C contents in plasma and blood are summarized Downloaded from in Table 2. After a single oral dose of 5 mg/177 nCi of [14C]cerlapirdine, 14C was determined in selected timepoints (up to 96 h) in blood and for all timepoints (up to 336 dmd.aspetjournals.org 14 h) in plasma (Figure 3). Mean peak concentrations (Cmax) of C were achieved with mean tmax values of ~ 3 h and ~ 6 h in blood and plasma, respectively. Mean Cmax values of 14C were 69.2 ng cerlapirdine equivalents/ml in blood and 104 ng eq/ml in plasma. at ASPET Journals on September 26, 2021

14 Mean t½ of C was 37.0 h in blood and 90.0 h in plasma. It should be noted that the apparent t½ in blood was shorter relative to plasma. This is probably due to the limited

(up to 96 h only) analysis of blood samples, such that the terminal phase was not completely characterized. AUCinf was 3216 ng eq•h/ml in blood and 5610 ng eq•h/ml in plasma. Over the 96-hour sampling period, mean blood/plasma ratios for 14C ranged between 0.59:1 (1 hour) and 0.89:1 (12 hours) post-dose for all subjects, suggesting that

[14C]cerlapirdine or related metabolites did not preferentially partition in whole blood components.

The pharmacokinetic parameters of cerlapirdine and desmethylcerlapirdine are also summarized in Table 2. The plasma concentrations vs. time profiles are depicted in

DMD Fast Forward. Published on September 12, 2014 as DOI: 10.1124/dmd.114.059675 This article has not been copyedited and formatted. The final version may differ from this version.

DMD #59675

Figure 4. Following a single oral 5 mg dose of [14C]cerlapirdine, cerlapirdine was absorbed into the systemic circulation with median time to peak plasma concentrations

(tmax) of ~3 h (range 1.5 to 6 h). Mean Cmax and AUCinf for cerlapirdine was ~82 ng/ml and 2866 ng•h/ml, respectively. Mean Vz/F and and mean CL/F were 2.1 l/kg and 0.024 l/h/kg, respectively, with mean apparent t½ of ~60 h. For desmethylcerlapirdine, median tmax value was ~13 h (range 4 to 48 h). Mean Cmax and AUCinf values for desmethylcerlapirdine were 5.0 ng/ml and 507 ng•h/ml, respectively. Downloaded from

Desmethylcerlapirdine was eliminated from the systemic circulation with a mean apparent t½ of ~85 h. Cerlapirdine and desmethylcerlapirdine accounted for 51% and 9%, dmd.aspetjournals.org respectively, of the total 14C in plasma. The ratio of desmethylcerlapirdine to cerlapirdine concentrations in plasma, based on their respective AUCinf values, was ~0.18.

at ASPET Journals on September 26, 2021

In Vitro Cytochrome P450 Reaction Phenotyping

In human liver microsomes, the substrate depletion or formation of desmethylcerlapirdine was inhibited minimally by the various CYP inhibitors evaluated with the exception of quercetin (48-55%) and ketoconazole (54-63%). These observations suggested that

CYP2C8 and CYP3A were primarily responsible for the metabolism of cerlapirdine to desmethylcerlapirdine, apparently with similar contribution of these enzymes in human liver microsomes. Since quercetin also inhibits CYP3A (albeit to a lower extent), the contribution of CYP2C8 to desmethylcerlapirdine formation may have been somewhat overestimated. Recombinant CYP2C8 and CYP3A4 were also shown to be primarily responsible for the metabolism of cerlapirdine to desmethylcerlapirdine, with Vmax values of 3.4 and 9.4 pmol/pmol CYP/min for CYP2C8 and CYP3A4, respectively, and Km

DMD Fast Forward. Published on September 12, 2014 as DOI: 10.1124/dmd.114.059675 This article has not been copyedited and formatted. The final version may differ from this version.

DMD #59675 values of 3.3 and 11.2 µM for CYP2C8 and CYP3A4, respectively. When corrected by their corresponding RAF values, the intrinsic clearance (Vmax/Km) values of desmethylcerlapirdine formation were 0.34 and 0.036 ml/mg/min for CYP2C8 and

CYP3A4, respectively, reflecting a corresponding contribution of ~90% for CYP2C8 by

~10% by CYP3A4. By taking into consideration results from both the recombinant CYPs and chemical inhibition in human liver microsomes, it appeared that the contribution of

CYP2C8 and CYP3A to desmethylcerlapirdine formation was 48-90% and 10-63%, Downloaded from respectively.

dmd.aspetjournals.org The reason behind the apparent discrepancy between the chemical inhibition method and the rCYP method on the contribution assessed for CYP2C8 and CYP3A4 is not clear, therefore, a range for their respective contributions was reported. The inter-individual at ASPET Journals on September 26, 2021 variability in the systemic exposure of cerlapirdine in the present study is relatively low

(25-30% CV), which suggests that CYP3A4 may not play a major role in the clearance of cerlapirdine. CYP2C8 genetic polymorphisms have been reported where the reduced function alleles CYP2C8*2 (found only in African-Americans with an allele frequency of

0.18) and CYP2C8*3 (occurred primarily in Caucasians with an allele frequency of 0.13) may potentially lead to variability in pharmacokinetics, clinical response and toxicity

(Dai et al., 2001). However, the size of the present study (n=6 subjects) is likely insufficient to detect such occurrences if they indeed exist.

Metabolite Profiling and Identification in plasma, urine and feces

DMD Fast Forward. Published on September 12, 2014 as DOI: 10.1124/dmd.114.059675 This article has not been copyedited and formatted. The final version may differ from this version.

DMD #59675

The radiochromatogram from the 0-72 h AUC plasma pool is depicted in Figure 5. The major peak (~92% of total sample radioactivity) co-eluted with cerlapirdine. One other peak was observed, co-eluting with desmethylcerlapirdine. No other metabolite was identified in the plasma pooled sample.

The amounts of 14C associated with peaks observed in the radiochromatograms obtained for pooled urine and feces samples are summarized in Table 3, and the metabolite Downloaded from profiles are depicted in Figures 6 and 7. In the pooled 0-96h urine sample (Figure 6), the major peak present (7.1% of the administered dose) co-eluted with cerlapirdine-N-oxide dmd.aspetjournals.org (M3). A second peak co-eluted with cerlapirdine (4.9% of the administered dose), and another with desmethylcerlapirdine (M1; 3.1% of the administered dose). A fourth peak, representing 2.6% of the administered dose, did not align with any of the available at ASPET Journals on September 26, 2021 reference standards. In the 96-168h pooled urine sample (data not shown), desmethylcerlapirdine, cerlapirdine-N-oxide, the unknown peak and cerlapirdine were observed, representing 1.5, 1.1, 0.6 and 0.7% of the administered dose, respectively. In the 0-96h pooled feces homogenate radio-chromatograms (Figure 7), cerlapirdine and desmethylcerlapirdine peaks were observed, with the parent compound showing higher abundance (24.5% of the administered dose) compared to the metabolite (14.7%) in the earlier time point sample and the metabolite showing higher abundance (5.4% compared to 3.4%) in the later time point (96-168 fecal pooled sample, data not shown).

Cerlapirdine-N-oxide or any other metabolite peak besides cerlapirdine and M1 was not observed in either the 0-96h or the 96-168h feces homogenate sample pools.

DMD Fast Forward. Published on September 12, 2014 as DOI: 10.1124/dmd.114.059675 This article has not been copyedited and formatted. The final version may differ from this version.

DMD #59675

Metabolite structures in the collected metabolite peaks from plasma, urine and feces homogenate sample pools were further confirmed by comparing the collision-induced mass spectra and with high resolution mass spectra of the metabolites to the authentic reference standards. Representative exact mass spectra for cerlapirdine, desmethylcerlapirdine and cerlapirdine-N-oxide observed in fractions collected from the

0-96h urine sample pool and the corresponding authentic standards are shown in Figure

8. Similar results were observed in fractions from plasma, fecal homogenates and the 96- Downloaded from

168h urine sample pools. For cerlapirdine, the parent ion at 410.15413 (2.1 ppm) possesses the proper exact mass protonated molecular ion. The main fragment ions at m/z dmd.aspetjournals.org 365.09621 (2.1 ppm) represents the loss of dimethylamine, while fragments at 219.02271

(C10H7N2O2S; 2.0 ppm) and 173.07123 (C10H9N2O; 1.68 ppm) represent fragmentation around both the dimethylamino and sulfone moieties (Figure 8A). The at ASPET Journals on September 26, 2021 desmethylcerlapirdine (M1) peak, which elutes on the LC column slightly ahead of the parent compound, shows a parent ion at m/z 396.13779 and is consistent with an empirical formula of C21H22N3O3S (0.38 ppm). The fragment ions at 365.09582,

219.02249 and 173.07098 are the same as for the parent drug (Figure 8B). Cerlapirdine-

N-oxide, M3, was observed only in urine. The mass spectrum of metabolite in urine fraction collected at a retention time aligned with the N-oxide shows a protonated molecular ion of m/z 426.14812, which is consistent with an empirical formula of

C22H24N3O4S (-0.20 ppm). The later retention time than parent is consistent with an N- oxide metabolite on the dimethylamino nitrogen, however, the daughter spectrum was not informative.

DMD Fast Forward. Published on September 12, 2014 as DOI: 10.1124/dmd.114.059675 This article has not been copyedited and formatted. The final version may differ from this version.

DMD #59675

The pooled urine radio-chromatogram shows a small peak not associated with parent drug, desmethyl or N-oxide metabolites at a retention time of approximately 37.5 min, which may suggest the presence of another metabolite. This peak accounted for ~10% and 15% of the total 14C recovered for the 0-96h and the 96-168h sample pools, respectively. However, the mass spectral data did not offer enough information to propose a structure for this material.

Downloaded from

DISCUSSION

Cerlapirdine is a 5-HT6 receptor antagonist that has been evaluated as a potential dmd.aspetjournals.org treatment for cognitive deficits in mild-to-moderate Alzheimer’s Disease (Brisard et al,

2010). As part of the clinical development program, a human ADME study with

[14C]cerlapirdine was conducted to elucidate the metabolism and disposition of at ASPET Journals on September 26, 2021 cerlapirdine. Due to the long retention time of cerlapirdine in pigmented tissues, a low radioactive dose (≤200 nCi) was administered. Due to the low amount of radioactivity that is generally below the detection limit of conventional detection method such as liquid scintillation counting, accelerator mass spectrometry was used to determine the 14C drug-related material in the plasma, urine and fecal samples obtained from this study.

Overall results show that cerlapirdine is well absorbed (at least 70% based on amounts of unchanged cerlapirdine in feces and amounts of metabolites excreted in urine and feces) after oral administration. Recovery of 14C was almost complete (mean total recovery was

98.3%) within 14 days, with feces being the major route of excretion, accounted for

~70% of the total dose, and urinary excretion was a minor route, with mean excretion of

DMD Fast Forward. Published on September 12, 2014 as DOI: 10.1124/dmd.114.059675 This article has not been copyedited and formatted. The final version may differ from this version.

DMD #59675

~28% of the dose. The rate of excretion was rapid, with majority (~68%) of the dose recovered within 96 h.

Metabolite profiling results showed that unchanged cerlapirdine was the major drug- related compound in circulation, accounting for approximately 51% of the total 14C in plasma. One circulating metabolite, desmethylcerlapirdine (M1), was identified. The ratio of desmethylcerlapirdine relative to cerlapirdine was 0.18 (based on AUCinf values). Downloaded from

Total desmethylcerlapirdine was approximately 9% of the total 14C in plasma.

Evaluation of the partial AUC data between 0 and 72h also shows that cerlapirdine and dmd.aspetjournals.org 14 M1 AUC0-72h values were approximately 80% of the C AUC0-72h in plasma (data not shown), indicating that the parent compound and M1 accounted for a majority of the total drug-related materials, even though the overall AUC values show that the parent and M1 at ASPET Journals on September 26, 2021 metabolite accounted for ~60% of total 14C AUC (0-336h). This discrepancy is primarily due to low levels of 14C in plasma samples beyond 72 h (concentrations <15 ng eq/mL).

The difference between the total 14C AUC and the measured cerlapirdine and M1 may be due to other minor metabolites (with abundance lower than desmethylcerlapirdine since no definitive peak has been observed) in plasma. The recovery of total 14C from pooled plasma was ~72%, which was similar to the extraction recoveries of cerlapirdine and desmethylcerlapirdine from plasma when evaluated by an LC/MS-MS assay (data on file). It is possible that some of the minor metabolites of cerlapirdine may not be quantitatively extracted and therefore not identified in this study. In vitro metabolism studies in human liver microsomes have shown that other oxidative metabolites (besides

M1 and the N-oxide, M3) are formed (data on file). Other oxidative metabolites have also

DMD Fast Forward. Published on September 12, 2014 as DOI: 10.1124/dmd.114.059675 This article has not been copyedited and formatted. The final version may differ from this version.

DMD #59675 been observed in metabolism studies in nonclinical species in vitro and in vivo (data on file). However, in earlier metabolite profiling studies after single or multiple doses of cerlapirdine in humans, cerlapirdine and desmethylcerlaperidine were the major drug- related materials in circulation, whereas cerlapirdine N-oxide was observed in trace amount after multiple dosing only (data on file). It is possible that low levels of these other oxidative metabolites (observed in human liver microsomes in vitro and in nonclinical species) may be present, but the abundance of these would be relatively low Downloaded from compared to cerlapirdine and desmethylcerlapirdine, since no definitive metabolite peak was observed in either the single/multiple dose metabolite profiling studies or in this dmd.aspetjournals.org study. If present, the abundance of these metabolites would be relatively low compared to cerlapirdine and desmethylcerlapirdine. It should also be noted that no other metabolites besides cerlapirdine, M1 and M3 has been observed in the pooled urine and fecal samples at ASPET Journals on September 26, 2021 from later timepoints (96-168 h), therefore, the presence of another metabolite at later time points (beyond 72 h) is not likely.

The N-oxide metabolite (M3) was the major drug-related entity excreted in urine, along with desmethylcerlapirdine and unchanged cerlapirdine. Unchanged cerlapirdine was the predominant compound excreted in the feces, followed by desmethylcerlapirdine. No other metabolite was identified in the feces. CYP2C8 and CYP3A were identified as the major CYP isozymes responsible for the formation of desmethylcerlapirdine in human liver microsomes or using recombinant enzymes. Both the desmethyl and the N-oxide metabolites were also identified from in vitro systems using human liver microsomes or

DMD Fast Forward. Published on September 12, 2014 as DOI: 10.1124/dmd.114.059675 This article has not been copyedited and formatted. The final version may differ from this version.

DMD #59675

S9 fractions (data on file). The overall metabolism and disposition pathways of cerlapirdine is summarized and depicted in Figure 9.

The AMS technique has been utilized in this metabolism study with satisfactory outcome.

However, samples are pooled across timepoints and subjects due to low amounts of radioactivity and 14C, therefore, time-related changes in the metabolite profiles and variability between subjects cannot be assessed. It should be noted that the metabolism of Downloaded from cerlapirdine is uncomplicated, with 1 major (desmethyl) and 1 minor (N-oxide) metabolite identified in plasma and in excreta. Other minor metabolites, if present, would dmd.aspetjournals.org likely occur at very low amounts precluding sufficient characterization and identification.

Since its introduction, accelerator mass spectrometry (AMS) has increasingly been at ASPET Journals on September 26, 2021 utilized in pharmaceutical research and development (Lappin and Stevens, 2008; Garner,

2010; Lappin and Seymour, 2010). The ultrahigh sensitivity of AMS (by measure isotopic 14C rather than radioactivity) enables the use of low amounts of radioactivity in radiotracer pharmacokinetic and metabolism studies. This is particularly important when the compound under evaluation shows radiolytic instability, has to be administered as very low dose, or the compound exhibits long systemic and/or tissue half-lives such that the total amount of radioactivity that can be safety administered is limited. In a conventional human ADME study utilizing liquid scintillation counting as a quantitation method, the typical radioactivity doses are in the range of 10 to 100 µCi. However, in the case of ixabepilone which shows radiolytic instability at high specific activity but was stable at 1-2 nCi/mg (Comezoğlu et al., 2009), AMS provided a tool by which the mass

DMD Fast Forward. Published on September 12, 2014 as DOI: 10.1124/dmd.114.059675 This article has not been copyedited and formatted. The final version may differ from this version.

DMD #59675 balance and metabolism of this compound could be determined in humans. Other examples of utilizing AMS for human mass balance and metabolism studies include vismodegib (Graham et al., 2011), which has a long half-life (over 200 h) and pasireotide, which is a somatostatin analog administered at a low dose of 600 µg (Lin et al., 2013).

Due to the low amount of radioactivity administered in studies utilizing AMS as the detection method, the radiotracer study may be exempt from regulatory review by health authorities, since the radioactive exposure from the administered dose is comparable to Downloaded from ambient exposure. As a result, PK/ADME study can be conducted during early phase clinical development, and valuable insight can be gained and resulted in an expedited dmd.aspetjournals.org development program (Garner et al., 2002; Garner 2010). This is particularly important in the case of identifying potential human metabolites that may require additional safety testing in animals and additional monitoring in clinical studies (Lappin and Seymour, at ASPET Journals on September 26, 2021

2010). In the case of cerlapirdine, long retention in pigmented tissues was observed in rodent tissue distribution studies (data on file), therefore, the radioactivity dose was limited to <200 nCi. Despite the low amount of radioactivity, the recovery of total 14C was virtually complete, while the metabolic and elimination pathways for cerlapirdine have also been delineated in this study.

Authorship Contributions:

Participated in research design: Susanna Tse, Louis Leung, Sangeeta Raje, Mark

Seymour

Conducted experiments: Louis Leung, Yoko Shishikura, R. Scott Obach

DMD Fast Forward. Published on September 12, 2014 as DOI: 10.1124/dmd.114.059675 This article has not been copyedited and formatted. The final version may differ from this version.

DMD #59675

Performed data analysis: Susanna Tse, Louis Leung, Sangeeta Raje, Mark Seymour,

Yoko Shishikura, R. Scott Obach

Wrote or contributed to the writing of the manuscript: Susanna Tse, Louis Leung,

Sangeeta Raje, Mark Seymour, Yoko Shishikura, R. Scott Obach Downloaded from dmd.aspetjournals.org at ASPET Journals on September 26, 2021

DMD Fast Forward. Published on September 12, 2014 as DOI: 10.1124/dmd.114.059675 This article has not been copyedited and formatted. The final version may differ from this version.

DMD #59675

REFERENCES

Arnt J, Bang-Andersen B, Grayson B, Bymaster FP, Cohen MP, DeLapp NW, Giethlen

B, Kreilgaard M, McKinzie DL, Neill JC, Nelson DL, Nielsen SM, Poulsen MN, Schaus

JM and Witten LM (2010) Lu AE58054, a 5-HT6 antagonist, reverses cognitive impairment induced by subchronic phencyclidine in a novel object recognition test in rats. Int J Neuropsychopharmacol 13: 1021-1033.

Downloaded from

Baird-Bellaire SJ, Wan H, Vandenhende F, Plotka A, Poola N, Danjou P and Chalon S

(2010) The effects of a single dose administration of SAM-531, a 5-HT6 antagonist, on dmd.aspetjournals.org sleep electroencephalogram (EEG) and quantitative wake EEG (QEEG) in healthy subjects. Ahzheimers Dement 6(4) Suppl: S510.

at ASPET Journals on September 26, 2021

Brisard C, Safirstein B, Booth K, Hua L, Brault Y, Raje S and Laventer S (2010) Safety, tolerability, and preliminary efficacy of SAM-531, a 5HT6 antagonist, in subjects with mild-to-moderate Alzheimer’s Disease: results from a phase 2A study. Ahzheimers

Dement 6(4) Suppl: S311.

Burgess PW, Quayle A and Frith CD (2001) Brain regions involved in prospective memory as determined by positron emission tomography. Neuropsychologia 39: 545-

555.

Codony X, Vela JM and Ramirez MJ (2011) 5-HT6 receptor and cognition. Curr Opin

Pharmacol 11: 94-100.

DMD Fast Forward. Published on September 12, 2014 as DOI: 10.1124/dmd.114.059675 This article has not been copyedited and formatted. The final version may differ from this version.

DMD #59675

Comery TA, Aschmies S, Haydar S, Hughes Z, Huselton C, Kowal D, Kramer A,

McFarlane G, Monaphan M, Smith D, Zhang G, Zhang Jm Zhang M-Y, Reinhart P,

Robichaud AJ and Pangalos MN (2010) SAM-531, N,N-dimethyl-3-{[3-(1- naphthylsulfonyl)-1H-indazol-5-yl]oxy}propan-1-amine, a novel serotonin-6 receptor antagonist with preclinical pro-cognitive efficacy. Ahzheimers Dement 6(4) Suppl: S548-

S549. Downloaded from

Cömezoğlu SN, Ly VT, Zhang D, Humphreys WG, Bonacorsi SJ, Everett DW, Cohen dmd.aspetjournals.org MB, Gan J, Beumer JH, Beijnen JH, Schellens JHM and Lappin G (2009)

Biotransformation profiling of [14C] ixabepilone in human plasma, urine and feces samples using accelerator mass spectrometry (AMS). Drug Metab Pharmacokinet 24(6): at ASPET Journals on September 26, 2021

511-522.

Dai D, Zeldin DC, Blaisdell JA, Chanas B, Coulter SJ, Ghanayem BI and Goldstein JA

(2001) Polymorphisms in human CYP2C8 decrease metabolism of the anticancer drug paclitaxel and arachidonic acid. Pharmacogenetics 11:597-607.

Dawson LA, Nguyen HQ and Li P (2000) In vivo effects of the 5-HT6 antagonist SB-

271046 on striatal and frontal cortex extracellular concentrations of noradrenaline, dopamine, 5-HT, glutamate and aspartate. Br J Pharmacol 130: 23-26.

DMD Fast Forward. Published on September 12, 2014 as DOI: 10.1124/dmd.114.059675 This article has not been copyedited and formatted. The final version may differ from this version.

DMD #59675

Foley AG, Murphy KJ, Hirst WD, Gallagher HC, Hagan JJ, Upton N, Walsh FS and

Regan CM (2004) The 5-HT6 receptor antagonist SB-271046 reverses scopolamine- disrupted consolidation of a passive avoidance task and ameliorates spatial task deficits in aged rats. Neuropsychopharmacology 29: 93-100.

Garner RC (2000) Accelerator mass spectrometry in pharmaceutical research and development-a new ultrasensitive analytical method for isotope measurement. Curr Drug Downloaded from

Metab 1(2): 205-213.

dmd.aspetjournals.org Garner RC (2010) Practical experience of using human microdosing with AMS analysis to obtain early human drug metabolism and PK data. Bioanalysis 2(3): 429-440.

at ASPET Journals on September 26, 2021

Garner RC, Goris I, Laenen AAE, Vanhoutte E, Meuldermans W, Gregory S, Garner JV,

Leong D, Whattam M, Calam A and Snel CAW (2002) Evaluation of accelerator mass spectrometry in a human mass balance and pharmacokinetic study-experience with 14C- labeled (R)-6-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]-4-(3- chlorophenyl)-1-methyl-2(1H)-quinilinone (R115777), a farnesyl transferase inhibitor.

Drug Metab Dispos 30: 823-830.

Geldenhuys WJ and Van der Schyf CJ (2009) The serotonin 5-HT6 receptor: a viable drug target for treating cognitive deficits in Alzheimer’s disease. Expert Rev Neurother

9(7): 1073-1085.

DMD Fast Forward. Published on September 12, 2014 as DOI: 10.1124/dmd.114.059675 This article has not been copyedited and formatted. The final version may differ from this version.

DMD #59675

Graham RA, Lum BL, Morrison G, Chang I, Jorga K, Dean B, Shin YG, Yue Q, Mulder

T, Malhi V, Xie M, Low JA and Hop CECA (2011) A single dose mass balance study of the Hedgehog pathway inhibitor vismodegib (GDC-0449) in humans using accelerator mass spectrometry. Drug Metab Dispos 39: 1460-1467.

Hah SS (2009) Recent advances in biomedical applications of accelerator mass spectrometry. J Biomed Sci 16: 54 doi: 10.1186/1423-0127-16-54. Downloaded from

Hamilton RA, Garnett WR and Kline BJ (1981) Determination of mean valproic acid dmd.aspetjournals.org serum level by assay of a single pooled sample. Clin Pharmacol Ther 29(3): 408-413.

Hirst WD, Abrahamsen B, Blaney FE, Calver AR, Aloj L, Price GW and Medhurst AD at ASPET Journals on September 26, 2021

(2003) Differences in the central nervous system distribution and pharmacology of the mouse 5-hydroxyltryptamine-6 receptor compared with rat and human receptors investigated by radioligand binding, site-directed mutagenesis, and molecular modeling.

Mol Pharmacol 64(6): 1295-1308.

Hoyer D, Clarke DE, Fozard JR, Hartig PR, Martin GR, Mylecharane EJ, Saxena PR and

Humphrey PPA (1994) International Union of Pharmacology classification of receptors for 5-hydroxytrytamine (serotonin). Pharmacol Rev 46(2): 157-203.

DMD Fast Forward. Published on September 12, 2014 as DOI: 10.1124/dmd.114.059675 This article has not been copyedited and formatted. The final version may differ from this version.

DMD #59675

Johnson CN, Ahmed M and Miller ND (2008) 5-HT6 receptor antagonists: prospects for the treatment of cognitive disorders including dementia. Curr Opin Drug Discov Devel

11(5): 642-54.

Lappin G and Seymour M (2010) Addressing metabolite safety during first-in-man studies using 14C-labeled drug and accelerator mass spectrometry. Bioanalysis 2(7):

1315-1324. Downloaded from

Lappin G and Stevens L (2008) Biomedical accelerator mass spectrometry: recent dmd.aspetjournals.org applications in metabolism and pharmacokinetics. Exp Opin Drug Metab Toxicol 4(8):

1021-1033.

at ASPET Journals on September 26, 2021

Lieben CKJ, Blokland A, Sik A, Sung E, van Nieuwenhuizen P and Schreiber R (2005) the selective 5-HT6 receptor antagonist Ro4368554 restores memory performance in cholinergic and serotonergic models of memory deficiency in the rat.

Neuropsychopharmacology 30: 2169-2179.

Lin TH, Hu K, Flarakos J, Sharr-McMahon M, Mangold JB, He H and Wang Y (2013)

Assessment of the absorption, metabolism and excretion of [14C]pasireotide in healthy volunteers using accelerator mass spectrometry. Cancer Chemother Pharmacol 72:181-

188.

DMD Fast Forward. Published on September 12, 2014 as DOI: 10.1124/dmd.114.059675 This article has not been copyedited and formatted. The final version may differ from this version.

DMD #59675

Lindner MD, Hodges Jr DB, Hogan JB, Orie AF, Corsa JA, Barten DM, Polson C,

Robertson BJ, Guss VL, Gillman KW, Starrett Jr JE and Gribkoff VK (2003) An assessment of the effects of serotonin 6 (5-HT6) receptor antagonists in rodent models of learning. J Pharmacol Exp Ther 307: 682-691.

Liu KG and Robichaud AJ (2009) 5-HT6 antagonists as potential treatment for cognitive dysfunction. Drug Dev Res 70: 145-168. Downloaded from

Liu KG and Robichaud AJ (2010) 5-HT6 medicinal chemistry. Int Rev Nuerobiol 94: 1- dmd.aspetjournals.org 34.

Maher-Edwards G, Dixon R, Hunter J, Gold M, Hopton G, Jacobs G, Hunter J and at ASPET Journals on September 26, 2021

Williams P (2011) SB-742457 and donepezil in Alzheimer disease: a randomized, placebo-controlled study. Int J Geriatr Psychiatry 26(6): 536-544.

Maher-Edwards G, Zvartau-Hind M, Hunter AJ, Gold M, Hopton G, Jacobs G, Davy M and Williams P (2010) Double-blind, controlled phase II study of a 5-HT6 receptor antagonist, SB-742457, in Alzheimer’s Disease. Curr Alzheimer Res 7: 374-385.

Marazziti D, Baroni S, Pirone A, Giannaccini G, Betti L, Schmid L, Vatteroni E, Palego

L, Borsini F, Bordi F, Piano I, Gargini C, Castagna M, Catena-Dell’Osso M and

Lucacchini A (2012) Distribution of serotonin receptor of type 6 (5-HT6) in human brain

DMD Fast Forward. Published on September 12, 2014 as DOI: 10.1124/dmd.114.059675 This article has not been copyedited and formatted. The final version may differ from this version.

DMD #59675 post-mortem. A pharmacology, autoradiography and immunohistochemistry study.

Neurochem Res 37: 920-927.

Marcos B, Gil-Bea FJ, Hirst WD, Garcia-Alloza M and Ramirez MJ (2006) Lack of localization of 5-HT6 receptors on cholinergic neurons: implication of multiple neurotransmitter systems in 5-HT6 receptor-mediated acetylcholine release. Eur J

Neurosci 24: 1299-1306. Downloaded from

Mitchell ES (2011) 5-HT6 receptor ligand as antidementia drugs. Int Rev Neurobiol 96: dmd.aspetjournals.org 167-187.

Mohler EG, Baker PM, Gannon KS, Jones SS, Shacham S, Sweeney JA and Ragozzino at ASPET Journals on September 26, 2021

ME (2012) The effects of PRX-07034, a novel 5-HT6 antagonist, on cognitive flexibility and working memory in rats. Psychopharmacology 220: 687-696.

Nakajima M, Tane K, Nakamura S, Shimada N, Yamazaki H and Yokoi T (2002)

Evaluation of approach to predict the contribution of multiple cytochrome P450s in Drug

Metabolism using relative activity factor: Effects of the differences in expression levels of NADPH-cytochrome P450 reductase and cytochrome b5 in the expression system and the differences in the marker activities. J Pharm Sci 91:952-963.

DMD Fast Forward. Published on September 12, 2014 as DOI: 10.1124/dmd.114.059675 This article has not been copyedited and formatted. The final version may differ from this version.

DMD #59675

Nyberg L, McIntosh AR, Cabeza R, Habib R, Houle S and Tulving E (1996) General and specific brain regions involved in encoding and retrieval of events: what, where and when. Proc Natl Acad Sci USA (93): 11280-11285.

Reimer C, Borroni E, Levet-Trafit B, Martin JR, Poli S, Porter RH and Bös M (2003)

Influence of the 5-HT6 receptor on acetylcholine release in the cortex: pharmacological characterization of 4-(2-bromo-6-pyrrolidin-1-ylpyridine-4-sulfonyl)phenylamine, a Downloaded from potent and selective 5-HT6 receptor antagonist. J Med Chem 46: 1273-1276.

dmd.aspetjournals.org Reinvang I, Magnussen S, Greenlee MW and PG Larsson (1998) Electrophysiological localization of brain regions involved in perceptual memory. Exp Brain Res 123: 481-

484. at ASPET Journals on September 26, 2021

Roberts JC, Reavill C, East SZ, Harrison PJ, Patel S, Routledge C and Leslie RA (2002)

The distribution of 5-HT6 receptors in rat brain: an autoradiographic binding study using

125 the radiolabelled 5-HT6 receptor antagonist [ I]SB-258585. Brain Res 934: 49-57.

Rosse G and Schaffhauser H (2010) 5-HT6 receptor antagonists as potential therapeutics for cognitive impairment. Curr Top Med Chem 10(2): 207-221.

Roth BL, Hanizavareh SM and Blum AE (2004) Serotonin receptors represent highly favorable molecular targets for cognitive enhancement in schizophrenia and other disorders. Psychopharmacology 174: 17-24.

DMD Fast Forward. Published on September 12, 2014 as DOI: 10.1124/dmd.114.059675 This article has not been copyedited and formatted. The final version may differ from this version.

DMD #59675

Salehpour M, Possnert G, Bryhni H (2008) Zeptomole sensitivity in biological accelerator mass spectrometry. Anal Chem 80(10): 3515-3521.

Sarapa N, Hsyu P-H, Lappin G and Garner RC (2005) The application of accelerator mass spectrometry to absolute bioavailability studies in humans: simultaneous administration of an intravenous microdose of 14C-nelfinavir mesylate solution and oral Downloaded from nelfinavir to healthy volunteers. J Clin Pharmcol 45: 1198-1205.

dmd.aspetjournals.org Schreiber R, Sleight A and Woolley M (2006) 5-HT receptors as targets for the treatment of cognitive deficits in schizophrenia, in The Serotonin Receptors: From Molecular

Pharmacology to Human Therapeutics (Roth BL ed.) pp 495-515, Humana Press, at ASPET Journals on September 26, 2021

Totowa, New Jersey.

Upton N, Chuang TT, Hunter AJ and Virley DJ (2008) 5-HT6 receptor antagonists as novel cognitive enhancing agents for Alzheimer’s Disease. Neurotherapeutics 5: 458-

469.

DMD Fast Forward. Published on September 12, 2014 as DOI: 10.1124/dmd.114.059675 This article has not been copyedited and formatted. The final version may differ from this version.

DMD #59675

Figure Legends

Figure 1. Structure of [14C]cerlapirdine (as hydrochloride salt) and the position of the 14C label.

Figure 2. Cumulative recoveries (mean±SD) of 14C (urine, feces and total) after a single, oral 5 mg (177 nCi) dose of [14C]cerlapirdine. Downloaded from

Figure 3. Plasma concentrations (mean ±SD) vs. time profiles of 14C in blood and plasma dmd.aspetjournals.org after a single, oral dose 5 mg (177 nCi) dose of [14C]cerlapirdine.

Figure 4. Plasma concentrations (mean ±SD) vs. time profiles of cerlapirdine and at ASPET Journals on September 26, 2021 desmethylcerlapirdine after a single, oral dose 5 mg (177 nCi) dose of [14C]cerlapirdine.

Figure 5. Metabolite profiles in 0-72 h plasma pooled sample after a single, oral 5 mg

(177 nCi) dose of [14C]cerlapirdine.

Figure 6. Metabolite profiles in 0-96 h urine pooled sample after a single, oral 5 mg (177 nCi) dose of [14C]cerlapirdine.

Figure 7. Metabolite profiles in 0-96 h pooled fecal homogenate pooled sample after a single, oral 5 mg (177 nCi) dose of [14C]cerlapirdine.

DMD Fast Forward. Published on September 12, 2014 as DOI: 10.1124/dmd.114.059675 This article has not been copyedited and formatted. The final version may differ from this version.

DMD #59675

Figure 8. HPLC/UV/HRMS-MS analysis of cerlapirdine, M1 and M3 in fractions obtained from 0-96h urine sample. (A) Top panel: UV chromatograms of fractions containing celapirdine, desmethylcelapirdine (M1), and celapirdine-N-oxide (M3). (B)

Lower panel: High resolution mass spectra for celapirdine, M1 and M3 metabolites.

Figure 9. Proposed metabolic and elimination pathways of cerlapirdine, and the amounts

(expressed as % of dose administered) of cerlapirdine, desmethylcerlapirdine and Downloaded from cerlapirdine-N-oxide excreted in urine and feces over 168 h.

dmd.aspetjournals.org at ASPET Journals on September 26, 2021

DMD Fast Forward. Published on September 12, 2014 as DOI: 10.1124/dmd.114.059675 This article has not been copyedited and formatted. The final version may differ from this version.

DMD #59675

Table 1. Demographic and Baseline Characteristics of Study Subjects.

Characteristics Mean (SD) Min-Max (Median), n=6

Age (year) 27.0 (10.7) 19.0-48.0 (24.5)

Baseline Height (cm) 183.17 (4.62) 178.0-189.0 (182.5)

Baseline Weight (kg) 76.8 (8.86) 66.3-89.0 (78.8)

Body Mass Index 22.98 (3.36) 18.76-28.09 (22.65) Downloaded from

Gender and Ethnicity

Sex Male (100%) dmd.aspetjournals.org Race Caucasian (100%)

at ASPET Journals on September 26, 2021

DMD #59675

Table 2. Pharmacokinetic parameters for total 14C in plasma and whole blood, cerlapirdine and desmethylcerlapirdine in

14 plasma following administration of a single oral dose of 5 mg (177 nCi) of [ C]cirlapirdine to healthy male subjects. This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. DMD FastForward.PublishedonSeptember12,2014asDOI:10.1124/dmd.114.059675

Plasma 14C (n=6) Blood 14C (n=6) Cerlapirdine (n=6) Desmethylcerlapirdine (n=6) Mean Min- Mean Min- Mean Min- Geometric Mean Min- Geometric Median Median (%CV) Max (%CV) Max (%CV) Max Mean (%CV) Max Mean C 104 71.3- 69.2 58.5- 81.8 50.6- 5.00 3.00- max 99.0 64.2 78.1 4.58 (ng/ml) (30) 122 (22) 92.4 (34) 127 (50) 9.27 5.7 4.0- 4.0 3.0- 3.42 1.5- 12.7 t (h) 6.0 3.0 3.15 4-48 7.78 max (27) 8.0 (39) 6.0 (44) 6.0 (137)

AUCt 5266 4443- 2607 1847- 2835 2022- 473 299- 5191 2595 2745 446 (ng•h/ml) (26) 7266 (26) 3751 (29) 4163 (39) 771

AUCinf 5610 3608- 3216 2283- 2866 2048- 507 341- 5475 3149 2778 482 (ng•h/ml) (24) 7568 (25) 4472 (28) 4187 (36) 791 79.4- 37.0a 29.5- 59.6 51.4- 84.6 57.8- t (h) 90 (11) 87.8 36.4 59.1 83.0 ½ 106 (14) 45.5 (14) 72.2 (22) 114 CL/F 0.024 0.02- NC NA NA NC NA NA 0.024 NA NA NA (l/h/kg) (21) 0.03 V /F 2.07 1.48- z NC NA NA NC NA NA 2.01 NA NA NA (l/kg) (28) 2.92 AUC inf NA NA NA NA NA NA 0.51 NA NA 0.09 NA NA Ratiob NC: Not calculated; NA: Not applicable a: The apparent t½ in blood may not reflect the true terminal phase, since blood samples were not analyzed beyond 96 h 14 b: AUCinf ratio relative to total C in plasma

Downloaded from from Downloaded dmd.aspetjournals.org at ASPET Journals on September 26, 2021 26, September on Journals ASPET at DMD #59675

Table 3. Amounts (expressed as % of dose administered) of cerlapirdine and metabolites excreted (0-336 h) in urine and feces

14 following administration of a single oral dose of 5 mg (177 nCi) of [ C]cerlapirdine to healthy male subjects. This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. DMD FastForward.PublishedonSeptember12,2014asDOI:10.1124/dmd.114.059675

Excreta Time Period (h) Cerlapirdine Desmethylcerlapirdine M3 Unassigneda Totalb

Urine 0-96 4.9 3.1 7.1 6.7 21.8

96-168 0.6 1.5 1.1 0.8 4.0

0-168 5.5 4.6 8.2 7.5 25.8

Feces 0-96 24.5 14.7 ND 6.6 45.8

96-168 3.4 5.4 ND 3.3 12.1

0-168 27.9 20.1 ND 9.9 57.9

Total 0-168 33.4 24.7 8.2 17.4 83.7 a: Minor peaks of 14C in urine and feces homogenate pooled samples; structures could not be identified b; Additional 2.2% excreted in urine pooled sample and 12.5% excreted in feces homogenate pooled sample between 168 and 336h

Downloaded from from Downloaded dmd.aspetjournals.org at ASPET Journals on September 26, 2021 26, September on Journals ASPET at DMD Fast Forward. Published on September 12, 2014 as DOI: 10.1124/dmd.114.059675 This article has not been copyedited and formatted. The final version may differ from this version.

Figure 1

Downloaded from

dmd.aspetjournals.org at ASPET Journals on September 26, 2021 DMD Fast Forward. Published on September 12, 2014 as DOI: 10.1124/dmd.114.059675 This article has not been copyedited and formatted. The final version may differ from this version.

Figure 2

120 Urine Feces 100 Total

60 Downloaded from

40 % Cumulative Dose % Cumulative

20 dmd.aspetjournals.org

0 0 48 96 144 192 240 288 336 Time, Hour at ASPET Journals on September 26, 2021 DMD Fast Forward. Published on September 12, 2014 as DOI: 10.1124/dmd.114.059675 This article has not been copyedited and formatted. The final version may differ from this version.

Figure 3

1000

Blood Plasma 100 Downloaded from

10 C Concentration, ng eq/ml C Concentration, ng 14 dmd.aspetjournals.org

1 -24 12 48 84 120 156 192 228 264 300 336 Time, hour

at ASPET Journals on September 26, 2021 DMD Fast Forward. Published on September 12, 2014 as DOI: 10.1124/dmd.114.059675 This article has not been copyedited and formatted. The final version may differ from this version.

Figure 4

100 Cerlapirdine Desmethylcerlapirdine

10 Downloaded from

1 Concentration, ng/ml dmd.aspetjournals.org

0.1 -24 12 48 84 120 156 192 228 264 300 336 Time, hour at ASPET Journals on September 26, 2021 DMD Fast Forward. Published on September 12, 2014 as DOI: 10.1124/dmd.114.059675 This article has not been copyedited and formatted. The final version may differ from this version.

Figure 5

Cerlapirdine

1000

750 Downloaded from 500 C (ng eq•h/ml) 14 M1 250 dmd.aspetjournals.org

0 20 30 40 50 Retention Time (min) at ASPET Journals on September 26, 2021 DMD Fast Forward. Published on September 12, 2014 as DOI: 10.1124/dmd.114.059675 This article has not been copyedited and formatted. The final version may differ from this version.

Figure 6

3 M3

Cerlapirdine 2 M1 Downloaded from

C (% C (% dose) administered 1 14 dmd.aspetjournals.org

Figure 7

Cerlapirdine 10 M1

5 Downloaded from

14C (% 14C (% administered dose) M3 dmd.aspetjournals.org

0 20 30 40 50 Retention Time (min) at ASPET Journals on September 26, 2021 Figure 8 (A) RT: 0.00 - 50.00 32.13 NL 6.00E4 60000 nm=309.5-: 310.5 PDA 40000 cerlapirdine 20100727- 20000 05

0 This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. 60000 NL:6.00E4

nm=309.5- DMD FastForward.PublishedonSeptember12,2014asDOI:10.1124/dmd.114.059675 40000 31.55 310.5 PDA 20100727- uAU 20000 desmethylcerlapirdine 04 0 60000 NL:6.00E4 33.09 nm=309.5 40000 -310.5 PDA 20000 20100731- cerlapirdine-N-oxide 01 0 0 5 10 15 20 25 30 35 40 45 50 Time (min)

(B)

Figure 9

cerlapirdine: urine 5.5%, feces 27.9%

CYP2C8,

CYP3A4 Downloaded from dmd.aspetjournals.org at ASPET Journals on September 26, 2021 M1 (desmethylcerlapirdine): M3 (cerlapirdine-N-oxide): urine 4.6%, feces 20.1% urine 8.3%