DMD Fast Forward. Published on July 9, 2010 as DOI: 10.1124/dmd.110.033092 DMDThis Fast article Forward. has not been Published copyedited and on formatted. July 9, The2010 final as version doi:10.1124/dmd.110.033092 may differ from this version. DMD/2010/033092

METABOLIC DISPOSITION OF CASOPITANT,

A POTENT NK1 RECEPTOR ANTAGONIST, IN MICE,

RATS AND DOGS

Authors:

Lidia Miraglia, Sabrina Pagliarusco, Ellenia Bordini, Silvia Martinucci and Mario Downloaded from

Pellegatti

Department of Drug Metabolism and Pharmacokinetics, Medicine Research

Center, GlaxoSmithKline, Verona, Italy dmd.aspetjournals.org at ASPET Journals on September 27, 2021

Copyright 2010 by the American Society for Pharmacology1 and Experimental Therapeutics. DMD Fast Forward. Published on July 9, 2010 as DOI: 10.1124/dmd.110.033092 This article has not been copyedited and formatted. The final version may differ from this version. DMD/2010/033092

RUNNING TITLE a) Metabolic disposition of casopitant in preclinical species. b) Lidia Miraglia, PCD DMPK GlaxoSmithKline, Via Fleming 4 37135 Verona, tel 0039 0458218465, fax 0039 0458218072, [email protected] c) Number of text pages: 34

Number of Tables: 6

Number of Figures: 6 Downloaded from Number of References: 27

Number of words in Abstract: 232 words

Number of words in Introduction: 309 words dmd.aspetjournals.org

Number of words in Discussion: 1050 words d) Abbreviations used are:

NK-1 = neurokinin-1 at ASPET Journals on September 27, 2021

SP = Substance P

GW679769 = 1-piperidinecarboxamide,4-(4-acetyl-1-piperazinyl)-N-

((1R)-1-(3,5-bis(trifluoromethyl)phenyl)-ethyl)-2-(4-fluoro-2-methylphenyl)-N- methyl-(2R,4S)

SD = Sprague-Dawley

CD-1 = Cluster of differentiation 1

BDC = bile duct-cannulated

HPLC = high performance liquid chromatography

LSC = Liquid scintillation counting

NMR = nuclear magnetic resonance

2 DMD Fast Forward. Published on July 9, 2010 as DOI: 10.1124/dmd.110.033092 This article has not been copyedited and formatted. The final version may differ from this version. DMD/2010/033092

ABSTRACT

Casopitant [1-piperidinecarboxamide,4-(4-acetyl-1-piperazinyl)-N-

((1R)-1-(3,5-bis(trifluoromethyl)phenyl)-ethyl)-2-(4-fluoro-2-methylphenyl)-N- methyl-(2R,4S)] is a potent and selective antagonist of the Neurokinin-1 (NK1) receptor, developed for the prevention of chemotherapy induced nausea and vomiting and post-operative nausea and vomiting. Absorption, distribution, metabolism and elimination of [14C]casopitant have been investigated in the Downloaded from mouse, rat and dog following single oral administration, and compared to human. [14C]casopitant was rapidly absorbed in all three species: maximum

plasma concentration of radioactivity was generally observed 0.5-2 hours after a dmd.aspetjournals.org single oral dose. In dog and female rat, as observed for man, the principal circulating radiolabelled components were unchanged casopitant and its hydroxylated derivative M13. In rats, there was an evident sex-related at ASPET Journals on September 27, 2021 difference in the rate of elimination of drug related material with elimination more rapid in males than females. In dogs and mice, no notable sex differences were observed in the pattern of excretion. The elimination of drug- related radioactivity was largely by metabolism, with metabolites excreted primarily in the feces. The predominant route of metabolism was the oxidation of the parent molecule, observed together with loss of the N-acetyl group, N- demethylation and modification of piperazine with consequent opening and cleavage of the ring, giving a complex pattern of metabolites. Conjugation of some of those oxidized products with glucuronic acid was observed. Urinary excretion in all three species was a minor route of elimination accounting between 2 and 7 % of the dose, with unchanged parent drug never quantifiable.

3 DMD Fast Forward. Published on July 9, 2010 as DOI: 10.1124/dmd.110.033092 This article has not been copyedited and formatted. The final version may differ from this version. DMD/2010/033092

Introduction

Neurokinin subtype-1 (NK-1) receptors are widely distributed in the peripheral and central nervous system including areas of the central nervous system thought to be involved in the vomiting reflex, such as the nucleus tractus solitarius (NTS) and the dorsal motor nucleus (DMN) of the vagus. They are activated by Substance P (SP), a neuropeptide member of the tachykinin family. SP and its NK-1 receptors are also found in non-neural tissues such as Downloaded from endothelial and inflammatory cells as well as gastrointestinal, respiratory, and genitourinary tissues. NK-1 receptor antagonists are believed to exert their anti-

emetic effect by blocking SP mediated receptor activation within the central dmd.aspetjournals.org vomiting center, which is essential for the processing of emetic stimuli and the coordination of the emetic reflex (Sanger et al., 2006; Diemunsch et al., 2009).

Casopitant, also known as GW679769, is a potent and selective at ASPET Journals on September 27, 2021 antagonist of the human NK-1 receptor both in vitro and in vivo and has good brain penetration properties.

Clinically, casopitant has been shown to be effective for the treatment of chemotherapy induced and post-operative nausea and vomiting (CINV and

PONV respectively) (Arpornwirat et al., 2006; Aziz et al. , 2008; Chung et al.,

2006; Grunberg et al. , 2008; Herrstedt et al. , 2008; Navari, 2008; Rolski et al.,

2006; Singla et al., 2006; Strausz et al., 2008).

The metabolism and excretion of casopitant in man have been reported recently

[Pellegatti et al., 2009]. In man, casopitant is well absorbed after oral administration and elimination is principally via the feces. It is extensively metabolized and only negligible amounts are excreted as unchanged compound. The two main circulating metabolites were the hydroxylated derivative M13 and the deacetylated and oxidized derivative M12.

4 DMD Fast Forward. Published on July 9, 2010 as DOI: 10.1124/dmd.110.033092 This article has not been copyedited and formatted. The final version may differ from this version. DMD/2010/033092

This article describes absorption, metabolism and elimination of casopitant in adult Sprague-Dawley (SD) rats, in CD-1 mice and in Beagle dogs after a single oral dose of [14C]casopitant (Fig. 1).

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 27, 2021

5 DMD Fast Forward. Published on July 9, 2010 as DOI: 10.1124/dmd.110.033092 This article has not been copyedited and formatted. The final version may differ from this version. DMD/2010/033092

MATERIALS AND METHODS

Chemicals

[14C]casopitant mesylate, [12C]casopitant mesylate, standards of metabolites M12 (coded as GSK631832), M13 (coded as GSK525060), M31

(coded as GSK517142), M44 (coded as GSK1735004) and M134 (coded as

GSK2110277), were all supplied by Chemical Development, GlaxoSmithKline.

The structures of metabolites are reported (Table 2). Downloaded from Radiolabelled [14C]casopitant was synthetised by GSK Isotope

Chemistry, UK: the specific activities were 0.37 MBq/mg for mouse study, 0.361

MBq/mg for rat and 0.074 MBq/mg for dog and the radiochemical purity was at dmd.aspetjournals.org least 99%. Commercially obtained chemicals and solvents were of HPLC or analytical grade. Liquid scintillation cocktails were obtained from Zinsser

Analytics (Frankfurt, Germany) and Perkin Elmer Life Science and Analytical at ASPET Journals on September 27, 2021

Instruments Inc (Waltham, MA).

Animals

Male and female Sprague Dawley (SD) rats (209-236 g for intact rats, and 202-281 g for BDC rats) were obtained from Charles River UK Ltd

(Margate, UK). Male (15-18 kg) and female (9-10 kg) Beagle dogs were taken from a colony at Charles River Laboratories (Edinburgh, UK). Male and female

CD-1 mice (21-36 g) were supplied by Charles River Laboratories (UK) and

Charles River (Lecco, IT).

The animals were kept under standard environmental conditions using routine methods of animal husbandry. Water from domestic supply and a standard diet were provided to animals. The animals were not fasted overnight

6 DMD Fast Forward. Published on July 9, 2010 as DOI: 10.1124/dmd.110.033092 This article has not been copyedited and formatted. The final version may differ from this version. DMD/2010/033092 but food was withheld at the time of dosing and was returned approximately 4 hours following dosing. Water was available ad libitum.

The rat study was conducted at Huntington Life Sciences (UK), the dog and mouse studies at Charles River Laboratories (UK) and at GlaxoSmithKline,

(Verona, IT).

All in life experiments described in this paper complied with national legislation and with the company policy on the care and use of laboratory Downloaded from animals and with related codes of practice.

Dose preparation and sample collection dmd.aspetjournals.org

The doses selected for rat and dog were the NOAELs during 13 and 4 week toxicity studies respectively, whilst for mouse it was the mean of the lowest doses used during toxicity studies up to 13 weeks of treatment. at ASPET Journals on September 27, 2021

For the total mass balance and metabolism studies, oral doses were prepared by dissolving an appropriate amount of [14C]casopitant mesylate in the appropriate volume of water for injections (with sonication as necessary). The formulation obtained was continually mixed using a magnetic stirrer until completion of dosing. Aliquots of the formulation were analysed by radio-HPLC before and after dosing, to determine the radiochemical purity and confirm the stability of [14C]casopitant in the dose formulation during both preparation and over the dosing period.

Mouse

Mice were housed singly in all glass metabolism cages specially designed for the separate, quantitative collection of urine and feces. Six male and six female mice received a single oral administration of [14C]casopitant

7 DMD Fast Forward. Published on July 9, 2010 as DOI: 10.1124/dmd.110.033092 This article has not been copyedited and formatted. The final version may differ from this version. DMD/2010/033092 mesylate at a target dose level of 50 mg free base/kg. Total radioactivity was determined in urine and feces collected up to 168 h post-dose. At the end of each excreta collection period, the cages were washed with water and acetonitrile as appropriate, and the wash retained for radioassay. Radioactivity was also determined in the carcasses at 168 h post dose. In addition, pre-dose excreta were collected to provide control matrices and storage stability samples.

Twenty five male and twenty five female mice were dosed at the same Downloaded from oral dose to investigate total radioactivity concentration in blood and plasma.

Five animals per sex were bled at each time-point. Blood collection was

performed by heart puncture prior to dosing and at 0.5, 2, 6 and 24 hours dmd.aspetjournals.org postdose under isoflurane anaesthesia. Blood samples were collected into tubes containing K3EDTA as anticoagulant. After removing aliquots for radioanalysis, the remaining blood samples were centrifuged at 2000 g for 10 at ASPET Journals on September 27, 2021 min at 4ºC to separate plasma samples.

Rat

The rats used were housed singly in all glass metabolism cages specifically designed for the separate, quantitative collection of urine, feces and

(where appropriate) bile. Three male and three female rats received a single oral dose of [14C]casopitant mesylate by gavage at 15 mg free base/kg. Urine and feces were collected at pre dose, and over 24 hour period up to 168 hours post dose. At 168 hours post dose, animals were killed by cervical dislocation under isoflurane anesthesia. Radioactivity was determined in the carcasses.

Seven days before dosing, four additional male and four female rats were surgically prepared with indwelling bile duct and duodenal cannulae under general anaesthesia (isoflurane) (Van Wijk et al., 2001). One end of a flexible

8 DMD Fast Forward. Published on July 9, 2010 as DOI: 10.1124/dmd.110.033092 This article has not been copyedited and formatted. The final version may differ from this version. DMD/2010/033092 cannula was inserted into the common bile duct of each rat and the other end inserted into the duodenum. The cannula loop was exteriorised, the incisions closed and a protective harness fitted. The animals were allowed to regain consciousness.. On the day prior to dosing, the cannula loop was opened.

The bile-duct cannulated (BDC) rats received a single oral dose of

[14C]casopitant at 15 mg free base/kg. After dosing the rats were infused with replacement bile salt solution (3.3 mg/mL cholic acid and 3.3 mg/mL taurocholic Downloaded from acid dissolved in a 5% solution of sodium bicarbonate in saline, adjusted to pH

8.0) through the duodenal cannula until the time of termination.

Urine and feces were collected prior to dosing and up to 96 hours post dmd.aspetjournals.org dose. Bile samples were collected overnight prior to dosing and during the periods 0-6 and 6-24 hours, then over 24 hours periods up to 96 hours after dosing. At the end of each excreta collection period the cages of both intact at ASPET Journals on September 27, 2021 and BDC rats were rinsed with water and water with acetonitrile as described before.

An additional three intact male and three intact female rats were exsanguinated under isoflurane anaesthesia and then killed by cervical dislocation at time points of 2, 8 and 24 hours post oral dosing (15 mg free base/kg). Blood samples were collected and plasma samples prepared as described before. Brains were also collected, rinsed with cold saline, weighed and then frozen on solid carbon dioxide.

Dog

The dogs used were housed in separate stainless steel metabolism cages. Urine and feces were collected from each of three male and two female dogs pre-dose, and over the period of 24 hours up to 216 hours. Blood

9 DMD Fast Forward. Published on July 9, 2010 as DOI: 10.1124/dmd.110.033092 This article has not been copyedited and formatted. The final version may differ from this version. DMD/2010/033092 samples were also collected (into tubes containing K3EDTA as anticoagulant) from the same animals at pre-dose and at 1, 6, 24 and 96 hours after single oral dose of [14C]casopitant mesylate at 10 mg free base/kg. Plasma was separated by centrifugation as described previously.

Urine and feces were also collected at pre dose and, at 24 hour intervals up to

96 hours from two additional male dogs that had been surgically prepared previously with indwelling bile duct cannulae, under general anaesthesia Downloaded from (isoflurane in oxygen/nitrous oxide). The common bile-duct had been cannulated with a T-piece catheter between the last hepatic junction and the

entrance to the duodenum. The T-piece catheter had three cannulae attached, dmd.aspetjournals.org which were connected to access ports implanted subcutaneously. Bile was allowed to flow continuously into the duodenum until the animals were required for dosing. A balloon at the end of the central cannula was inflated at ASPET Journals on September 27, 2021 approximately 2 h prior to dose administration to divert bile into the proximal cannula for collection via the access port (Kissinger et al., 1998).

These dogs were administered at the same dose as the previous ones.

Bile samples were also collected from each BDC dog pre-dose and over 0-6, 6-

24, 24-48, 48-72 and 72-96 hours post-dose. After dosing the dogs were infused with replacement bile salt solution (cholic acid [18 g] and sodium bicarbonate [1.3 g] in 1 L of 0.9% w/v aqueous sodium chloride solution, pH adjusted to 7.4 to 7.8).through the duodenal cannula until the time of termination.

At the end of each excreta collection period, the cages were washed with mixture of water and ethanol and the wash retained for radioassay.

Assay of total radioactivity

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Aliquots of urine, cage washes, bile and plasma samples were mixed with either Aquasafe 500 plus or Ultima Gold or Pico Fluor-40M scintillator.

Feces samples were weighed, an appropriate amount of water added and the total weight recorded prior to homogenisation. Aliquots of each homogenate were combusted using a Packard Tri-Carb 307 Automatic Sample

14 Oxidiser (PerkinElmer Life and Analytical Sciences). The resultant CO2 was collected by absorption in Carbosorb® (8 mL) to which Permafluor®E+ Downloaded from scintillation fluid (10 mL) was added.

Carcasses were solubilised in a caustic digestion solution containing

aqueous sodium hydroxide, methanol and Triton X-100. Aliquots of each digest dmd.aspetjournals.org were combusted as described above for fecal samples.

Brain samples were homogenised with water (1:2 w/w) on ice. Aliquots of each brain homogenate and rat, mouse and dog blood samples were at ASPET Journals on September 27, 2021 combusted as described previously.

Finally all samples were analysed by using Packard Tricarb 2100 TR liquid scintillation counter (PerkinElmer Life and Analytical Sciences), with automatic quench correction by an external standard method (Botta et al.,

1985).

Preparation of Plasma and Brain Samples for Radio-LC-MS/MS Analysis

Plasma samples as well as brain homogenate samples from individual animals were pooled to produce a single representative sample per time point per sex.

Sample pools were extracted by mixing aliquots with ca. 3 volumes of organic solvent (methanol for mouse and dog, a mixture of 50:50 acetonitrile/methanol for rat). After centrifugation, the supernatants were

11 DMD Fast Forward. Published on July 9, 2010 as DOI: 10.1124/dmd.110.033092 This article has not been copyedited and formatted. The final version may differ from this version. DMD/2010/033092 removed, the pellets re-suspended in the same solvent and volume and the process repeated up to three times. The supernatants were combined, evaporated under nitrogen and reconstituted in an appropriate volume of water/methanol or water/acetonitrile. Weighed aliquots of each extract were radioassayed by LSC for the calculation of recovery after extraction and reconstitution prior to radio-HPLC analysis.

Downloaded from Preparation of Excreta Samples for Radio-LC-MS/MS Analysis

Fecal homogenates, urine and bile samples (bile for rat and dog only) for

each animal were obtained by pooling across sampling times by total weight dmd.aspetjournals.org ratio in order to generate sample pools containing 90% or greater of the radioactivity excreted in the matrix.

For feces, sample pools were processed as described previously for at ASPET Journals on September 27, 2021 plasma and brain samples. Weighed aliquots of each extract were radioassayed by LSC for the calculation of recovery after extraction and reconstitution prior to HPLC analysis.

Urine and bile samples were centrifuged to remove any particulates, and analysed by LSC before and after centrifugation to determine whether any notable losses of radioactivity had occurred. Bile samples were diluted 1:5 with water prior to radio-HPLC analysis.

Quantification and profiling of metabolites

Due to the large number of chemically different metabolites a variety of HPLC methods were employed as described below.

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HPLC method 1 (used for analysis of rat and dog plasma, rat brain,

mouse, rat and dog feces samples)

The chromatographic instrument used consisted of an Agilent 1100 binary pump, autosampler and column oven (50oC) (Agilent Technologies, Palo Alto,

CA) using a Phenomenex (Torrance, CA) Synergi Polar RP column (250 x 4.6 mm, 4 μm). The mobile phase consisted of 5 mM ammonium formate aq. (pH 5 adjusted with formic acid) (solvent A) and 5 mM ammonium formate aq. (pH 5) Downloaded from in acetonitrile (10:90 water/acetonitrile) (solvent B) at a flow rate of 1 mL/min. A gradient was used, starting at 37% B with a linear change to 57% B over 60

minutes, followed by a linear increase to 100% by 65 minutes, with these dmd.aspetjournals.org conditions being maintained for a further 7 minutes. For female rat feces, after

60 minutes the gradient was followed by two linear increases: to 80% by 75 minutes and to 100% by 75.1, with these conditions being maintained for a at ASPET Journals on September 27, 2021 further 10 minutes.

HPLC method 2 (used for analysis of mouse plasma samples)

The chromatographic instrument used consisted of two HPLC pumps 305 and

306 (Gilson Inc, Middleton, WI), Waters 717 autosampler (Milford, MA) and

Waters TCM (Milford, MA) column oven (35oC) using a Phenomenex

(Torrance, CA) Synergi Hydro RP column (150 x 4.6 mm, 4 μm). The mobile phase consisted of 2.5 mM ammonium acetate (unadjusted pH) (solvent A) and acetonitrile (solvent B) at a flow rate of 1 mL/min. A gradient was used, starting at 5% B with a linear change to 80% B over 50 minutes, followed by a linear increase to 90% by 50.1 minutes, with these conditions being maintained for a further 5 minutes.

HPLC method 3 (used for analysis of rat and dog bile samples)

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The chromatographic method is as described for method 1 with the exception of the gradient, which started at 10% of B with a linear increase to 37% over 10 minutes, followed by further three linear increases: to 43% B by 40 minutes, to

60% B by 65 minutes and then to 100% B by 75 minutes. These conditions were maintained for a further 5 minutes.

For the analysis of urine samples another HPLC method, not reported here, was used. Downloaded from HPLC column recoveries were determined on selected samples by collecting the total HPLC column eluate for the appropriate run and assaying

the radioactivity to assess recovery of injected radioactivity. Full recoveries of dmd.aspetjournals.org radioactivity were obtained from the HPLC eluate collected (> 96%).

Radio-HPLC data were captured off line (Bruin et al., 2006): chromatographic fractions were collected using a Perkin Elmer fraction collector at ASPET Journals on September 27, 2021 mod IntekServices FC LH200 into 96 deep well microtitre plates

(LUMAPLATES) containing yttrium silicate solid scintillant (PerkinElmer Life and

Analytical Sciences). Radioactivity determination was performed by scintillation counting (TopCount NXT counter, PerkinElmer Life and Analytical Sciences).

Radio-HPLC data from mouse feces samples only were captured on line using a Packard Flo-One Beta 500TR detector (PerkinElmer Life and Analytical

Sciences) with a liquid scintillant.

Structural identification of metabolites

Structural characterization was performed on selected samples by

Radio-HPLC-Mass Spectrometry using a hybrid quadrupole/time-of-flight Q-

TOF Ultima (Waters MS Technologies, Manchester, UK) tandem mass spectrometer (Morris et al., 1996). Electrospray ionization, in positive and/or

14 DMD Fast Forward. Published on July 9, 2010 as DOI: 10.1124/dmd.110.033092 This article has not been copyedited and formatted. The final version may differ from this version. DMD/2010/033092 negative mode, was used. The HPLC flow was split (1:5) between mass spectrometer and a fraction collector model FC 204 (Gilson Inc, Middleton, WI), which was used for off-line radiodetection.

Metabolites were identified based on charged molecular ions, mass accuracy and their collision induced dissociation fragmentation (Oliveira et al.,

2000). Authentic standards, when available, were used to compare chromatographic retention times and fragmentation patterns. For some Downloaded from metabolites a definitive structure had been previously confirmed by 1H-NMR

(Plumb et al., 1999) in clinical studies (Pellegatti et al., 2009).

Fully characterized metabolites were designated by the letter M followed dmd.aspetjournals.org by a number; where a synthetic standard was available a GSK code number was assigned. at ASPET Journals on September 27, 2021

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RESULTS

Plasma and blood radioactivity concentrations in mice, rats and dogs

After a single oral administration of [14C]casopitant to CD-1 mice

(50 mg/kg), the highest levels of drug related material in plasma were detected at 2 hours in males and 0.5 hours in females. Blood and plasma levels of radioactivity were comparable between males and females with blood/plasma ratios ranging between 0.7 and 0.9. Downloaded from Following a single oral administration of [14C]casopitant (15 mg/kg) to SD rats, maximum mean concentrations of radioactivity in blood and plasma were

observed at 2 hours and 8 hours post dose in males and females, respectively. dmd.aspetjournals.org

Concentrations in these matrices were greater in females than males at corresponding time points by up to 4-fold. Generally the circulating radioactivity was evenly distributed between the plasma and blood cellular fractions. at ASPET Journals on September 27, 2021

In Beagle dogs, after single oral administration of [14C]casopitant

(10 mg/kg), circulating radioactivity was predominantly associated with the plasma fraction. The mean concentration of drug related material in plasma was equivalent between sexes with the highest level reached at 1 hour post dose.

Brain radioactivity concentration in rats

Following a single oral dose of [14C]casopitant (15 mg/kg) to SD rats, maximum mean concentrations of radioactivity in brain were observed at 2 hours and 8 hours post dose in males and in females respectively. Mean brain:plasma concentration ranged between 0.2 and 0.4 and between 0.7 and

1.2 in males and females, respectively, up to 24 hours post dose.

Total mass balance

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Recovery of radioactivity in mice, rats and dogs following single oral administration of [14C]casopitant is shown in Table 1.

Excretion in mice

After a single oral dose of [14C]casopitant to CD-1 mice at 50 mg/kg, there were no sex differences in the routes and rates of elimination of radioactivity. The major route of elimination was via the feces (about 86% of the dose) with urinary excretion accounting for a mean of about 6%. Elimination Downloaded from of drug related material was rapid in both sexes, with >85% of the administered dose recovered by 24 hours. The mean total recoveries were >99%.

dmd.aspetjournals.org

Excretion in rats

Following a single oral administration of [14C]casopitant (15 mg/kg) to intact SD rats, the major route of elimination of radioactivity was via the feces (a at ASPET Journals on September 27, 2021 mean of 97% of the dose). Urinary elimination was minor (a mean of 3 % of the dose). The mean total recovery of radioactivity was about 100% in both sexes.

In BDC rats, the main routes of elimination of radioactivity were via the bile (a mean of 57% and 38% of the dose in male and females, respectively) and feces (a mean of 33% and 52% of the dose in males and females, respectively). Urinary elimination was minor, accounting for less than 5% of the dose. A mean of at least 65% and 47% was absorbed by male and female rats, respectively, as judged by the radioactivity eliminated in the bile and urine, and

DRM found in the residual carcass. The mean total recovery of radioactivity was about 98% in both sexes.

There was a sex-related difference in the rate of elimination of drug related material with elimination of radioactivity in both intact and BDC animals being more rapid in males than females. In intact animals about 92% of the

17 DMD Fast Forward. Published on July 9, 2010 as DOI: 10.1124/dmd.110.033092 This article has not been copyedited and formatted. The final version may differ from this version. DMD/2010/033092 dose was recovered by 24 hours post dose in males, compared to only 37% in females.

Excretion in dogs

After a single oral administration of [14C]casopitant to intact beagle dogs

(10 mg/kg), the major route of elimination of radioactivity was via the feces

(about 84% of the dose). Urinary excretion was a minor route of elimination Downloaded from (about 6% of the dose). Elimination of radioactivity was initially rapid with a mean of about 80% of the administered radioactivity recovered in the excreta

and cage wash of either sex by 72 hours post dose. However, small amounts dmd.aspetjournals.org of radioactivity continued to be recovered, primarily in the feces up to 216 hours. The mean total recovery was about 91%.

In BDC dogs, there were notable differences in the extent of elimination at ASPET Journals on September 27, 2021 via the bile and feces between animals. In one animal the major route of elimination was via the feces (55%), with biliary excretion accounting for 25%, whereas in the second, the major route was via the bile (65%), with fecal excretion accounting for 26%.

Although inter individual variability in the biliary elimination is common in dogs and no issues have been reported during the surgical procedure, is possible that some technical and model-related problems have occurred that could explain the discrepancy between the two animals.

Metabolite profiles

Proposed metabolite structures and supporting spectral data for all matrices are shown in Table 2.

Plasma

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Unchanged casopitant was at least one of three principal radio-peaks observed in all species, but represented the major one at all time points only in female rat samples.

Mouse plasma metabolite profiles showed relevant differences from the other species: in male mouse the major metabolites were M28, resulting from cleavage of the molecule, and its oxidized derivatives, M16 and M17, ranging between 18% and 7% of plasma radioactivity at 0.5h and between 6% and 1% Downloaded from at 6h. Female plasma profiles were qualitatively similar to the male ones; therefore only male results are reported and discussed.

Plasma metabolite profiles were similar overall in female rat and male dmd.aspetjournals.org and female dog (female dog data not reported). The major circulating metabolites resulted from different modifications of the piperazine ring including the hydroxylated derivative M13, and the N-dealkylated piperazine derivatives at ASPET Journals on September 27, 2021

M31 and M134, ranging from 6% to 24% of radioactivity across all the time points. These components were also present in male rat plasma, although the principal circulating component in this matrix was M3; the methyl hydroxylated derivative, accounting for 20 to 39% of the plasma radioactivity. Other minor components observed in both species were the deacetylated and oxidized derivatives M9 and M12, the N-demethylated M69 and two oxidized metabolites

M10 and M109. Metabolites M57 and M155, N,N-deethylated hydroxylated derivatives, were detected in the dog only. Quantification for some relevant metabolites in plasma after oral administration at selected time points are summarized in Table 3 and representative radiochromatogram profiles are shown in Fig. 2 and Fig.3.

Brain

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The principal radiolabelled component in male and female rat brain extracts was unchanged parent representing ca. 50-80% of the brain radioactivity. Other notable radiolabelled components were M13 (in male and female) and M3 (in male only), representing ca 12-24% and 9% of the brain radioactivity, respectively. Fig. 4 shows representative radiochromatograms of male and female rat brain extracts. Quantitative results from brain samples are summarized in Table 4. Downloaded from Feces

Radio-HPLC analysis of male and female rat, dog and mouse fecal extracts

14 after oral administration of [ C]casopitant showed complex metabolic profiles, dmd.aspetjournals.org with many radiolabelled components, the majority of which did not represent more than 2% of the administered dose in all species.

Mean quantification data for fecal radiometabolites are detailed in Table at ASPET Journals on September 27, 2021

5. Metabolite profiles of all intact animals were qualitatively similar to BDC ones, moreover mouse and dog profiles were qualitatively similar between genders (data not shown). Therefore results from male intact animals only are presented, with the exception of rat, whose observed sex differences require some discussion on female rat results too.

In male mouse feces the principal radiolabeled components were multiple-oxidized metabolites M50 and M80 representing together ca. 29% of the administered dose. Other minor metabolites were observed including oxidized derivatives (M61, M63 and M137), cleavage metabolites such as M28 and M16 (oxidized M28 derivative) and N-dealkylated piperazine oxidized derivative M141, each representing no more than 4-8% of the administered dose.

20 DMD Fast Forward. Published on July 9, 2010 as DOI: 10.1124/dmd.110.033092 This article has not been copyedited and formatted. The final version may differ from this version. DMD/2010/033092

The principal radiolabeled derivatives in intact male rat feces, as for the mouse, were metabolites M50 and M80, representing together ca. 60% of the administered dose. Instead, in female rat the fecal radioactivity was distributed among several radio-peaks, the major of which included N-dealkylated piperazine derivatives M31, M134, M169, accounting together for ca 9% of the administered dose and the co-eluting metabolites M41 (oxidized derivative) and

M1 (a N-demethylated oxidized derivative), representing together ca. 7% of the Downloaded from administered dose. The remaining radio-components accounted individually in both genders for less than 5% of the administered dose. Fig. 5a shows a

representative radiochromatogram of female rat feces extract. dmd.aspetjournals.org

The principal radiolabeled compounds in male dog feces were derived from methyl hydroxylation (M3), and either oxidation on the piperazine ring followed by N-dealkylation (M31, M123, M134) or N-demethylation and further at ASPET Journals on September 27, 2021 oxidation (M33).

In all species unchanged parent was a minor component.

Bile

Radio-HPLC analysis of male and female rat and dog bile after oral administration of [14C]casopitant showed many radiolabeled components partially co-eluting. Results are summarized in Table 5.

Female and male rat bile radio-profiles were qualitatively similar, but individual radio-peaks differed in their relative abundances. In male rat bile several glucuronide oxidized derivatives were detected (M100, M102, M103,

M106, M118, M119, M171, M172, M173) accounting together for ca. 25% of the dose. Another notable radio-peak corresponded to co-eluting metabolites M50 and M81 (N-deacetylated hydroxylated glutathione conjugate), representing 9% of the dose. In female rat bile each radio-peak accounted for 3% or less of the

21 DMD Fast Forward. Published on July 9, 2010 as DOI: 10.1124/dmd.110.033092 This article has not been copyedited and formatted. The final version may differ from this version. DMD/2010/033092 administered dose. The major radio-components observed were several oxidized glucuronide derivatives (M78, M103, M104, M120) and a cysteine derivative (M122) together representing ca. 7% of the dose. N-demethylated oxidized derivatives (M1, M33, M63, M70) together accounted for another 6% of the dose. Fig. 5b shows a representative radiochromatogram of female rat bile.

Male and female dog bile metabolic profiles were qualitatively and quantitatively similar with several radio-peaks each accounting for less than 4% Downloaded from of the dose. Major metabolites observed included an oxidized derivative (M37),

N-deacetylated hydroxylated glutathione conjugates (M81, M82) and two

oxidized glucuronides (M103, M119). dmd.aspetjournals.org

In both rat and dog bile unchanged parent was detected only in negligible quantities.

Urine at ASPET Journals on September 27, 2021

Urine was a very minor route of excretion for all species; therefore result of intact and male animals only are presented. The quantifiable metabolites in the urine of all species were the cleavage metabolites M28 and a few of its further oxidized derivatives, including M16, M21, M22 and M138.

Non radiolabelled metabolites

Metabolites derived from the cleavage of [14C]casopitant with loss of the radiocarbon were also observed in the urine of all species by mass spectrometry only, identified as M114 and M115 (two oxidized derivatives), M44 and M136 (two glucuronide conjugates) and M117 (a sulfate conjugate). Some of them were also observed in plasma and bile of all species, as detailed in

Table 3 and Table 5.

22 DMD Fast Forward. Published on July 9, 2010 as DOI: 10.1124/dmd.110.033092 This article has not been copyedited and formatted. The final version may differ from this version. DMD/2010/033092

DISCUSSION

The absorption, metabolism and elimination of [14C]casopitant, after single oral administration at doses ranging from 10 to 50 mg/kg, have been fully characterized in mouse, rat and dog.

Casopitant was rapidly adsorbed in all three species, with maximum plasma concentration of radioactivity generally observed 0.5-2 hours after single oral dosing. The routes of excretion of the drug related material were similar in Downloaded from all preclinical species and also in man (Pellegatti et al., 2009). Elimination of casopitant occurred almost exclusively by metabolism (with almost two hundred

metabolites identified across the different species and matrices) and mainly via dmd.aspetjournals.org the feces, the matrix accounting for approximately 90% of the dose in mouse and rat and 80% in dog. Urine was a very minor route of elimination, representing in all species no more than 7% of the dose. The recovery of at ASPET Journals on September 27, 2021 radioactivity was ≥ 90% and for the most part was eliminated within 96h post dosing. Similar to man, in the dog the rate of elimination of radioactivity was slow, with small amounts of radioactivity excreted up to 216h.

Only rats showed an evident sex related difference in the rate of excretion of the drug related material, with elimination in both intact and BDC animals being more rapid in males than females. Sex-dependent differences in xenobiotic metabolism in rats can largely be attributed to differences in the profile of cytocrome P450 isoforms found in male and female liver and intestine

(Martignoni et al., 2006). In fact, the isoforms CYP3A2 and CYP3A9 in rat are respectively related to androgens and estrogen secretion, at least in liver if not in the intestine (Aiba et al., 2005), whereas CYP3A18 and CYP3A23 are constantly higher in male (Mahnke et al., 1997).

23 DMD Fast Forward. Published on July 9, 2010 as DOI: 10.1124/dmd.110.033092 This article has not been copyedited and formatted. The final version may differ from this version. DMD/2010/033092

The 10-30% less total cytocrome P450 expression in females as opposed to males (Mugford et al., 1998), could explain the slower casopitant metabolism observed in females, resulting in lower clearance and higher concentrations of the parent compound observed in both plasma and liver.

In addition, Phase II enzymes, such as glucuronyltransferases (Strasser et al., 1997) and glutathione S-transferases (Mugford et al., 1998), are sex dependant, often showing a higher activity in male than female rats. The Downloaded from increased biliary excretion of glucuronide, glutathione and cysteine derivatives of casopitant observed in BDC male rats (30% of the dose compared with only

7% in females) seems to agree with this result and with the hypothesis that dmd.aspetjournals.org estrogens cause decreases in the biliary excretion of xenobiotics in rat (Vore et al., 1980). The hydrophilicity of these conjugates derivatives may favor their elimination compared with the less polar casopitant unconjugated metabolites of at ASPET Journals on September 27, 2021 females, which tend rather to be retained and accumulated in the liver.

Few conjugate derivatives were present in feces, suggesting that hydrolysis for the most of metabolites occurred in the gastrointestinal tract.

The casopitant metabolism observed in female rat and dog showed the highest similarities with that found in man, which appeared to be mediated primarily through CYP3A4 (Pellegatti et al., 2009). In fact, the human isoform

CYP3A4 seems to exhibit some parallels when compared with the female rat dominant isoforms CYP3A9, -3A62 (Matsubara et al., 2004) and the canine

CYP3A12 and -3A26.

A summary of the observed metabolic pathways is reported in Table 6 and, based on the putative structure of metabolites a simplified metabolic scheme is shown in Figure 6.

24 DMD Fast Forward. Published on July 9, 2010 as DOI: 10.1124/dmd.110.033092 This article has not been copyedited and formatted. The final version may differ from this version. DMD/2010/033092

The major human route of metabolism, involving modifications of the piperazine moiety, such as oxidations, ring opening and N-dealkylation until loss of the ring, was principal also in the dog and female rat.

The multiple oxidations of the parent compound, mostly at the level of the piperidine and of the methyl on the fluorobenzene ring, were minor routes of metabolism in man but relevant in preclinical species, being observed primarily in rat and mouse. Downloaded from N-demethylation and N-deacetylation were observed to different extent in man and preclinical species, also in combination with extensive oxidation and

conjugation, leading to very complex metabolic profiles. The cleavage of the dmd.aspetjournals.org molecule with loss of the radiocarbon and its further conjugation was detected above all in the urine, probably because of its high polarity and lower molecular weight. However, with the exception of mouse, the cleavage was a very minor at ASPET Journals on September 27, 2021 pathway of metabolism in all species.

Because of the extensive number of detected metabolites, each of them in the excreta generally accounted for only small percentage of the dose, such as the unchanged casopitant present at negligible levels both in feces and urine.

Human, dog and female rat plasma profiles after single oral administration of casopitant were qualitatively equivalent. The two major

[definition in accordance with the European Medicine Agency (ICH Harmonized

Tripartite Guideline (M3) (R2), 2009)] human circulating metabolites after oral administration (Pellegatti et al., 2009), M13 and M12, were principal also in these two species. They both resulted from the oxidation of the piperazine moiety and were shown to be active in man. The other human active metabolite

M31, derived from N,N-piperazine deethylation, showed very high exposures in

25 DMD Fast Forward. Published on July 9, 2010 as DOI: 10.1124/dmd.110.033092 This article has not been copyedited and formatted. The final version may differ from this version. DMD/2010/033092 animals, but was present at very limited concentration in man. These three active human metabolites were observed in mouse and male rat too, but the major circulating metabolites in these species resulted respectively from the cleavage of the parent compound (M28 and M16) in the mouse and from hydroxylation of the methylbenzene in the male rat (M3).

Casopitant and the three active metabolites M13, M12 and M31, together with the very minor M69, were also the only DRM observed in both male and Downloaded from female rat brain.

In conclusion, consistent with the ex vivo data from man, elimination of

casopitant in preclinical species after single oral administration occurs almost dmd.aspetjournals.org exclusively by metabolism, with metabolites excreted primarily in the feces. The principal routes of metabolism involve loss of the N-acetyl and N-methyl groups, oxidations of either the methylbenzene or of the piperazine ring leading to at ASPET Journals on September 27, 2021 subsequent cleavage of this moiety, conjugation of various oxidized derivatives with glucuronic acid and several combinations of these pathways. The animal exposures of the most relevant circulating human metabolites are either comparable to or higher than those in the man, thus confirming the right choice was made when selecting the preclinical species during the safety assessment of casopitant. It can be considered that the safety profile of these metabolites has been fully investigated as part of the parent casopitant development program.

26 DMD Fast Forward. Published on July 9, 2010 as DOI: 10.1124/dmd.110.033092 This article has not been copyedited and formatted. The final version may differ from this version. DMD/2010/033092

ACKNOWLEDGMENTS

The authors would like to acknowledge Patrizia Fizzotti and Ivan Strepponi for technical and scientific assistance; Ken Lawrie for synthesizing the labeled compounds; GSK Chemical Development (Verona, IT) particularly Zadeo

Cimarosti for synthesizing standards of metabolites; Charles River Laboratories

(Edinburgh, UK) for conducting experimental activities; GSK Drug Metabolism and Pharmacokinetic department (Verona, IT), particularly Dimitri Colato, Downloaded from Patrizia Griffini, Mahmud Kajbaf, Dawn Osbourn, and Emilia Vaccaro for scientific support. dmd.aspetjournals.org at ASPET Journals on September 27, 2021

27 DMD Fast Forward. Published on July 9, 2010 as DOI: 10.1124/dmd.110.033092 This article has not been copyedited and formatted. The final version may differ from this version. DMD/2010/033092

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33 DMD Fast Forward. Published on July 9, 2010 as DOI: 10.1124/dmd.110.033092 This article has not been copyedited and formatted. The final version may differ from this version. DMD/2010/033092

LEGENDS FOR FIGURES

Figure 1:

Structure of [14C]casopitant

Figure 2:

Representative reconstructed radiochromatograms of pooled male (a) mouse and (b) dog plasma following a single oral administration of [14C]casopitant at a target dose of 50 mg and 10 mg free base/kg, respectively. Downloaded from Figure 3:

Representative reconstructed radiochromatograms of pooled (a) male and (b)

14 female rat plasma following a single oral administration of [ C]casopitant at a dmd.aspetjournals.org target dose of 15 mg free base/kg.

Figure 4:

Representative reconstructed radiochromatograms of pooled (a) male and (b) at ASPET Journals on September 27, 2021 female brain rat following a single oral administration of [14C]casopitant to

Sprague-Dawley rats at a target dose of 15 mg free base/kg

Figure 5:

Representative radiochromatogram of (a) feces and (b) bile following a single oral administration of [14C]casopitant to intact and bile duct cannulated female

Sprague-Dawley rats at a target dose level of 15 mg free base/kg

Figure 6:

Putative simplified metabolic scheme for casopitant in preclinical species

34 DMD/2010/033092

TABLES This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. TABLE 1 DMD FastForward.PublishedonJuly9,2010asDOI:10.1124/dmd.110.033092 Recovery of radioactivity in mice, rats and dogs following single oral administration of [14C]casopitant.

Mice (50 mg/kg) Rats (15 mg/kg) Dogs (10 mg/kg)

Matrixa Male Female Male Female Male Female

Intact Intact Intact BDC Intact BDC Intact BDC Intact

(n = 6) (n = 6) (n = 3) (n = 2) (n = 3) (n = 2) (n = 3) (n = 2) (n = 2)

Urine 5.4 (+/- 5.2) 6.9 (+/- 4.4) 3.4 (+/- 0.3) 4.6 1.5 (+/- 0.1) 1.7 5.9 (+/- 0.5) 5.4 5.8

Feces 84.9 (+/- 10.5) 86.7 (+/- 9.1) 96.4 (+/- 2.7) 33.3 97.6 (+/- 1.2) 51.6 82.5 (+/- 1.7) 25.5-55.4c 85.5

Bile ND ND ND 56.5 ND 38.1 ND 24.7-65.2c ND

Cage Wash 8.4 (+/- 6.5) 5.9 (+/- 7.2) 0.0 0.0 0.0 0.3 1.6 (+/- 1.0) 0.2 1.2

Carcass 0.4 (+/- 0.2) 0.7 (+/- 0.4) 0.4 (+/- 0) 3.5 2.1 (+/- 0.2) 6.9 ND ND ND

Totalb 99.2 (+/- 6.5) 100 (+/- 4.5) 101 (+/- 3.4) 97.9 102 (+/- 1.0) 98.9 90.1 (+/- 1.1) 85.8-96.2c 92.5

a Results are reported as mean unless stated otherwise and (+/- Standard Deviation)].

b Total collection period was 0-168 hr for mice and rats (intact), 0-96 hr for rats and dogs (BDC), 0-216 hr for dogs (intact).

ND = not determined

c individual values have been reported since there were notable differences in the extent of elimination between the two animals.

35

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Data are presented to one decimal place and are computer generated and rounded appropriately. As a consequence, calculations of This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. values yield minor deviations. DMD FastForward.PublishedonJuly9,2010asDOI:10.1124/dmd.110.033092

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TABLE 2 This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. Relevant metabolites of [14C]casopitant DMD FastForward.PublishedonJuly9,2010asDOI:10.1124/dmd.110.033092 1 Positive [M+H]+ or Negative [M+H]- H-NMR (600Mhz, 1:1 ACN:D2O)

Proposed Structure ion MS (where available)

Products Ions (m/z)

7.89 (brs, 1H), 7.59 (brs, 2H), 7.21

(dd, 1H), 6.88 (dd, 1H) 6.76 (dt, 1H),

O F + CH N FF 617 [M+H] 5.32 (q, 1H, NCHCH ), 4.23 (m, 1H), 3 3 N CH3 NN F F 489, 481, 346, 327, 298, 277, 241, 3.49 (m, 1H), 2.86 (m, 1H), 2.69 (s, CH O F 3 CH3 210, 167 3H, NCH ), 2.34 (s, 3H), 1.46 (d, 3H, F Casopitant 3

CHCH3), several piperidine and

piperazine signals v. broad.

O F

3CH N FF N H 619 [M+H]+ N N F OH F - CH O CH F 3 3 601, 467, 318, 190, 167, 336, 208 F M1

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7.90 (brs, 1H), 7.60 (brs, 2H), 7.25 This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion.

O F (dd, 1H), 7.07 (dd, 1H) 6.84 (dt, 1H), + 3CH N FF

633 [M+H] DMD FastForward.PublishedonJuly9,2010asDOI:10.1124/dmd.110.033092 N CH 3 5.35 (q, 1H), 4.74 and 4.64 (d, 1H N N F F 615, 505, 481, 369, 362, 241, 210, O F CH OH 3 each, CH2OH), 2.65 (s, 3H), 1.46 (d, 173, 167 F M3 3H), several signals broad and/or

obscured.

O F - NH FF N CH O 3 NN F 603 [M+H]+ F CH O CH F 3 3 489, 327, 298, 241, 175

F M9

O F - OH N FF N + CH tentative 3 619 [M+H] N N F F CH O CH F 3 3 489, 327, 298, 241, 175 F M10

- F NH FF + N 589 [M+H] O CH3 F NN F CH O CH F 3 3 489, 453, 249, 241, 182, 139

F GSK631832 (M12)

38

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7.88 (brs, 1H), 7.59 (brs, 2H), 7.16 This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion.

O F 633 [M+H]+ (m, 1H), 6.84 (dd, 1H) 6.72 (m, 1H), 3CH N FF N CH 3 DMD FastForward.PublishedonJuly9,2010asDOI:10.1124/dmd.110.033092 OH F NN F O F 615, 497, 489, 479, 461, 275, 183, 5.81 and obscured (m, 1H, rotamers, 3CH CH3

F GW525060 (M13) 165 CHOH) 5.35, several signals broad

and/or obscured.

O - 3CH N N CH 393 [M+H]+ 3 NNH OH CH O 3 336, 210, 208, 190, 167 F M16

O - 3CH N N CH3 391 [M+H]+ NNH or isomer/ O tautomer CH O 3 210, 190, 167 F M17

7.20 (dd, 1H), 6.92-6.98 (m, 2H) ,

+ O 393 [M+H] 5.74 and 5.39 (m, 1H, rotamers, 3CH N N OH CH 3 N NH + O MS/MS of [M+H] - H2O: 344, 275, CHOH), 2.49 (s, 3H), 2.34 (s, 3H) 3CH

F M21 239, 208, 165 several signals broad and/or

obscured.

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O - This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. 3CH N N O CH NNH 391 +

[M+H] DMD FastForward.PublishedonJuly9,2010asDOI:10.1124/dmd.110.033092 CH O 3 192, 184, 167

F M22

O 7.21 (dd, 1H), 6.92-6.97 (m, 2H) , CH N 3 + N CH 3 377 [M+H] N NH O 2.49 (s, 3H), 2.34 (s, 3H) several CH 3 241, 210, 167 F M28 signals broad and/or obscured.

O + F 591 [M+H] - 3CH N FF H NH CH3 N N F F O F 489, 455, 327, 320, 251, 241, 184, 3CH CH3

F GSK517142 (M31) 175, 141

O OH F - 3CH N FF N + H 635 [M+H] N N F OH F O F 3CH CH3 617, 465, 291, 245, 208, 165, 180 F M33

O + OH F 647 [M+H] - 3CH N FF N CH O 3 NN F F O F 489, 467, 327, 298, 263, 241, 197, 3CH CH3

F M37 153

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O + F 635 [M+H] - OH N

FF This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. N or isomers CH3 N N F OH F O F 617, 505, 487, 336, 327, 298, 272, 3CH CH3 DMD FastForward.PublishedonJuly9,2010asDOI:10.1124/dmd.110.033092 F M41 241, 190, 173, 131

7.91 (brs, 2H), 8.03 (brs, 1H), 5.18 F - FF 433 [M-H] Gluc O F (q, 1H) 4.09 (d, 1H, anomeric)1.45 F F CH 3 GSK1735004 (M44) 415, 241, 213, 175 (d, 3H)

O F - 3CH N FF N + CH3 647 [M+H] N N F OH O F CH O CH F 3 3 332, 272, 241, 210, 204, 167 F M50

O OH F - CH N FF 3 H NH + CH 3 607 [M+H] NN F F O F 3CH CH3 548, 489, 412, 327, 298, 241, 141 F M57

O OH F - CH N FF 3 + N 649 [M+H] CH3 N N F O OH F CH O CH F 3 3 631, 465, 348, 330, 305, 222, 208, 165 F M61

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O + F 633 [M+H] - 3CH N FF N This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. H N N F O F 467, 350, 332, 290, 222, 210, 204, O CH F OH 3 DMD FastForward.PublishedonJuly9,2010asDOI:10.1124/dmd.110.033092 F M63 167

O F - 3CH N FF N + H 603 [M+H] N N F F CH O CH F 3 3 475, 467, 346, 277, 241, 210, 167 F M69

O F - NH FF + N H 591 [M+H] NN F OH F CH O CH F 3 3 573, 308, 290, 208, 190, 182 F M70

F Gluc - (OH)2 FF + CH3 N N F 699 [M+H] F O F 3CH CH3 523, 505, 487, 371, 327, 298, 241 F

M78

O OH F - 3CH N FF N + CH 663 [M+H] 3 F OH N N O F O F CH CH 3 3 645, 330, 305, 241, 208, 204 F M80

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OH F - NH SG FF + This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. N 896 [M+H] CH3 NN F F O F 3CH CH3

767, 589, 571, 489, 453, 446, 308 DMD FastForward.PublishedonJuly9,2010asDOI:10.1124/dmd.110.033092 F M81, M82

823 [M+H]+ -

Gluc O F 805 (not for M118), 647, 519 (M100 3CH N FF N CH3 NN F OH O F O F only), 481 (not for M118), 332, 290 3CH CH3

F M100, M102, M106, (not for M118), 272 (M100 only),

M118 241(not for M118), 210, 204(not for M118), 167(not for M118)

O Gluc F - CH N 3 FF + N 809 [M+H] CH3 N N F F O F 3CH CH3 633, 615, 505, 481, 362, 210, 167 F

OH M103

+ F 683 [M+H] - OH FF

3CH NN F F O F 507, 489, 397, 379, 327, 298, 241, 3CH CH 3

Gluc F M104 175

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O O - F + This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. 3CH N FF N 633 [M+H] CH 3 NN F F O F 3CH CH 3 489, 392, 327, 298, 241, 163 DMD FastForward.PublishedonJuly9,2010asDOI:10.1124/dmd.110.033092 F M109

F - FF 287 [M-H]- OH F F F 269, 243, 223, 203, 195 OH O M114

F FF 257 [M-H]- - OH F F F CH 3 M115 213, 193, 173

F FF 337 [M-H]- - HO S 3 O F F F CH 3 M117 257, 237, 213, 97

Gluc + - O OH F 825 [M+H]

3CH N FF N CH3 N N F F 807, 649, 631, 613, 505, 479, 467, CH O F 3 CH3 OH F 291, 245, 226, 208, 183, 165 M119

Gluc Gluc F O - O O F OH NH FF CH N FF O 3 O + N H NH CH CH 797 [M+H] 3 F 3 N N NN F F F O F CH O F 3CH CH3 3 CH3 621, 184 F F or M120

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738 [M+H]+ - This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. Unknown cysteine conjugate M122 672, 489, 401, 327, 241, 184 DMD FastForward.PublishedonJuly9,2010asDOI:10.1124/dmd.110.033092 + OH F 564 [M+H] - CH O 3 FF NH CH3 NN F F O F 489, 428, 327, 315, 298, 272, 241, 3CH CH3

F M123 224, 175, 157, 114

F FF - NH + 2 CH 3 506 [M+H] N N F F O F 3CH CH3 370, 315, 298, 241, 175 F GSK2110277 (M134)

F a: 7.92 FF 449 [M-H]- b: 8.00 Gluc O F F F c: 5.04 273, 255, 243, 213, 193, 113 OH d: 3.80,3.91 M136 Gluc. anomeric: 4.45 7.91 (brs, 1H), 7.61 (brs, 2H), 7.26

O F (dd, 1H), 7.07 (dd, 1H) 6.84 (dt, 1H), NH FF N CH + 3 619 [M+H] N N F O F O F 5.35 (q, 1H), 4.74 and 4.66 (d, 1H CH OH 3 304, 286, 276, 272, 241, 204, 182 F M137 each, CH2OH), 2.62 (s, 3H), several

signals broad and/or obscured.

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+ O OH 393 [M+H] -

3CH N This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. N CH3 N NH + O MS/MS of [M+H] - H2O ion: 344, 275, 3CH

F M138 DMD FastForward.PublishedonJuly9,2010asDOI:10.1124/dmd.110.033092 239, 208, 165

O + F 621 [M+H] - 3CH N FF H NH CH3 N N F OH O F O F 519, 455, 350, 306, 290, 241, 204, 3CH CH3

F M141 184, 141

(O)2 - F + O OH NH FF F 607 [M+H] N CH N FF 3 H CH3 NH F CH NN 3 NN F F CH O CH F F 3 3 CH O CH F 489, 241, 200, 157 3 3

F or F M155

F + CH OH 550 [M+H] - 3 FF NH CH 3 NN F F 414, 327, 298, 279, 272, 241, 210, CH O F 3 CH3

F M169 184, 175, 143, 100

839 [M+H]+ -

Gluc O OH F 821, 803 (not for M171) 663 (M171 3CH N FF N CH 3 F OH N N O F O F only), 645 (M171 only), 479 (M173 3CH CH3

F M171 M172 M173 only), 330 (not for M172), 305(M171

only),: 272(M171 only),: 208: 165

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(M173 only) This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. The underlined metabolites in the table have been confirmed with either NMR or comparison with standards DMD FastForward.PublishedonJuly9,2010asDOI:10.1124/dmd.110.033092

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TABLE 3 This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. Percentage of radioactivity of casopitant and its relevant metabolites in mouse, rat and dog plasma after a single-dose oral DMD FastForward.PublishedonJuly9,2010asDOI:10.1124/dmd.110.033092 administration of [14C]casopitant

Peak ID % plasma radioactivity a

Mice (50 mg/kg) Rat (15 mg/kg) Dog (10 mg/kg)

male male female male

0.5 hr 6 hr 2 hr 8 hr 2 hr 8 hr 1 hr 6 hr

casopitant 12.2 6.5 13.2 3.9 45.4 43.1 25.6 12.0

M3 5.8 6.0 39.4 20.5 2.7 (+M166) 4.9 (+M166) BQL ND

M9 ND ND ND ND 1.7 (+M112) 1.2 (+M112) 5.6 4.2

M10 4.4 (+M69) 4.0(+M69) 1.4 1.2 1.6 1.3 5.3 4.0

M12+M69 ND ND 3.2 3.7 4.5 7.2 3.2 (+M154) 3.1(+M154)

M13 5.8 3.9 6.1 2.8 13.4 24.5 7.2 6.4

M16 13.5 3.9 ND ND ND ND ND ND

M17 7.0 1.1 ND ND ND ND ND ND

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M28 18.0 5.6 ND ND ND ND 3.5 3.2 This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion.

M31+M134 ND ND 4.3 6.1 8.9 7.3 11.7 16.8 DMD FastForward.PublishedonJuly9,2010asDOI:10.1124/dmd.110.033092 M57+M155 ND ND ND ND ND ND 3.4 5.7

M109 ND ND ND ND 10.1 0.8 4.3 (+M1) BQL(+M1)

Total μg eq/g in plasma 5.37 5.60 1.39 0.762 1.76 2.10 6.28 4.91

M44, M117 (male rat) M114, M115 (rat and dog), M136 (dog) were non-radiolabelled metabolites observed by mass spectrometry

only

a Observed metabolite radioactivity was determined by 96-well fraction collection with scintillation counting for 5 minutes after a chromatographic separation was performed by HPLC. Percentages obtained from the radio-chromatogram have been adjusted for the extraction recovery. The percentage of radioactivity per time point does not equal 100% since only distinct radioactive peaks were assigned values and only those above 5% of plasma radioactivity in at a least one specie were reported in the table.

BQL = below quantification limit, set to 25 cpm as peak area

ND = not detected

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DMD/2010/033092

TABLE 4

Percentage of radioactivity of casopitant and its relevant metabolites in male and

female rat brain after a single-dose oral administration of 15 mg/kg

[14C]casopitant

Peak ID % brain radioactivitya Downloaded from male female

2 hr 8 hr 2 hr 8 hr 24 hr

casopitant 66.5 50.7 81.6 75.4 50.0 dmd.aspetjournals.org

M3 9.1 BQL ND ND ND

M12+M69 BQL BQL 2.1 3.1 5.3

M13 11.9 BQL 9.4 11.5 23.8 at ASPET Journals on September 27, 2021

M31 BQL 2.0 ND

Total μg eq/g in brain 0.629 0.262 2.05 2.50 0.620

a Observed metabolite radioactivity was determined by 96-well fraction collection with scintillation counting for 5 minutes after a chromatographic separation was performed by

HPLC. Percentages obtained from the radio-chromatogram have been adjusted for the extraction recovery. The percentage of radioactivity per time point does not equal 100% since only distinct radioactive peaks were assigned values and a few minor metabolites were not reported.

BQL = below quantification limit, set to 25 cpm as peak area

ND = not detected

= Observed by HPLC mass spectrometry only

50 DMD Fast Forward. Published on July 9, 2010 as DOI: 10.1124/dmd.110.033092 This article has not been copyedited and formatted. The final version may differ from this version.

DMD/2010/033092

TABLE 5

Mean percentage of administered dose of casopitant and its relevant metabolites

in excreta following a single oral dose of [14C]casopitant to mice, rats and dogs

Peak ID % administered dosea

Mice (50 mg/kg) Rat (15 mg/kg) Dog (10 mg/kg)

male male female male Downloaded from Feces (intact)

casopitant 1.5 1.1 4.0 0.3

M1 ND 2.1 (+M41+M42) 6.8 (+M41) 2.5 (+M181) dmd.aspetjournals.org

M3 1.1 (+M9+M205) 0.9 2.7 3.2

M13 ND BQL 3.7 0.5

M28+M141 4.3 0.7 0.3 ND at ASPET Journals on September 27, 2021

M31+M134 0.6 (+M169) 1.3 (+M169) 9.0 (+M169) 3.3

M33 ND 1.0(+M164) 2.5 2.9

M50 22.2 51.3 2.3 ND

M61 6.2 (+M137) 0.8 1.2 (+M63) ND

M63 7.8 (+M16) 3.6 1.2 (+M61) ND

M76 ND BQL 4.6 (+M111+M170) ND

M80 7.1 10.4 (+M106) 1.1 ND

M123 ND 1.2 (+M62+M70) 3.5 (+M62+M70) 4.6

Total % dose in 84.9 96.4 97.6 82.5 feces Bile

casopitant NS BQL 0.5 0.4

51 DMD Fast Forward. Published on July 9, 2010 as DOI: 10.1124/dmd.110.033092 This article has not been copyedited and formatted. The final version may differ from this version.

DMD/2010/033092

Peak ID % administered dosea

Mice (50 mg/kg) Rat (15 mg/kg) Dog (10 mg/kg)

male male female male

M1+M42 NS 0.7 (+M41) 1.5 0.8

M3 NS 0.4 ND 1.7 (+M9+M36)

M12 NS BQL 0.5 (+M131) 1.2 Downloaded from M31+M134 NS BQL 0.9 1.6 (+M13+M57)

M33 NS 0.7 1.7 1.5

M37 NS ND ND 3.8(+M81+ M82) dmd.aspetjournals.org

M50 NS 9.0 (+M81) 0.7 ND

M63 NS 1.5 (+M78) 1.3 ND

M70 NS 0.8 (+M62+M123) 1.0 2.2 (+M123) at ASPET Journals on September 27, 2021

M78 NS 1.5 (+M63) 1.5 ND

M80 NS 3.7 (+M122+M174) 0.9 (+M81+M82) ND

M100+M172 NS 6.9 ND ND

M103 NS 9.6 (+M102+M106) 3.4 (+M104) 3.3

M104 NS 0.9 (+M79+M120) 3.4 (+M103) 1.9 (+M187)

M118+M171 NS 2.9 ND ND

M119 NS 5.2 (+M173) 1.0 2.1

M120 NS 0.9 (+M79+M104) 1.7 (+M122) ND

M122 NS 3.7 (+M80+M174) 1.7 (+M120) ND

M123 NS 0.8 (+M62+M70) 0.3 2.2 (+M70)

M174 NS 3.7 (+M80+M122) 0.3 ND

52 DMD Fast Forward. Published on July 9, 2010 as DOI: 10.1124/dmd.110.033092 This article has not been copyedited and formatted. The final version may differ from this version.

DMD/2010/033092

Peak ID % administered dosea

Mice (50 mg/kg) Rat (15 mg/kg) Dog (10 mg/kg)

male male female male

M185 NS ND ND 1.3

Total % dose in NS 56.5 38.1 45.0 bile Urine (intact) Downloaded from casopitant BQL BQL

M20 0.2 0.4 0.2 ND

M21 0.1 0.1 BQL 0.5(+M198) dmd.aspetjournals.org

M28 1.3 0.4(+M22) 0.2 1.3

M16 1.7 0.3 ND 0.4

M138 0.1 0.1 0.2 0.9 at ASPET Journals on September 27, 2021

Total % dose in 5.4 3.4 1.5 5.9 urine M44, M114, M115 and M136 were non-radiolabelled metabolites observed in all tree species by mass spectrometry only

a Percentages obtained from the radio-chromatogram have been adjusted for the

sample treatment recovery. The total percentage of the dose excreted in each matrix

does not equal to the sum of metabolite percentages, since not all the time points were

pooled and a few minor metabolites were not reported.

BQL = below quantification limit, set to 25 cpm as peak area

ND = not detected

NS = no sample

= Observed by HPLC mass spectrometry only.

53 DMD/2010/033092

TABLE 6 This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. Metabolic pathways observed in excreta of mice, rats and dogs following single oral administration of [14C]casopitant. DMD FastForward.PublishedonJuly9,2010asDOI:10.1124/dmd.110.033092 Piperazine ring Methyl Species N-demethylation N-dealkylation at N-deacetylation Glutathione opening & & piperazine Glucuronidation (gender) & Oxidation carbamate group & Oxidation conjugation Oxidation Oxidation

MICE M3, M61, M16, M20, M21, M141 M63 M137 (m) M50, M80 M28 M138

% dose 0-4 % 29-36 % 0-8 % 3-15 % 0-6 % NA

M100, M102,

M103, M106, RAT M31, M123, M41, M50, M16, M20, M21, M1, M33, M63 M118, M119, M81 (m) M134, M169 M80 M28 M138 M171, M172

M173

% dose 1-2 % 51-76 % 4-9 % 0- 1% 25-35 % 0-9 %

RAT M31, M123, M3, M13, M78, M103, M1, M33, M63 M20, M28, M138 M76 (f) M134, M169 M41, M50, M104 M119

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Piperazine ring Methyl Species N-demethylation N-dealkylation at N-deacetylation Glutathione This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. opening & & piperazine Glucuronidation

(gender) & Oxidation carbamate group & Oxidation conjugation DMD FastForward.PublishedonJuly9,2010asDOI:10.1124/dmd.110.033092 Oxidation Oxidation

M61,M80

% dose 9-17 % 10-20 % 6-16 % 0-1 % 0-5 % 5 %

DOG M31, M123, M16, M21 M103, M104, M3, M37 M1, M33 M9, M12 M81, M82 (m) M134, M169 M28,M138 M119

% dose 8-12 % 3-9 % 5-8 % 2-3 % 1-3 % 3-7 % 0-4 %

% of the dose figures are reported as a range due to metabolite co-elutions.

NA: Not Applicable, since mouse bile was not collected.

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