Metabolic Disposition of Casopitant, a Potent Neurokinin-1 Receptor Antagonist, in Mice, Rats, and Dogs

Transcription

1 /10/ $20.00 DRUG METABLISM AD DISPSITI Vol. 38, o. 10 Copyright 2010 by The American Society for Pharmacology and Experimental Therapeutics 33092/ DMD 38: , 2010 Printed in U.S.A. Metabolic Disposition of Casopitant, a Potent eurokinin-1 Receptor Antagonist, in Mice, Rats, and Dogs Lidia Miraglia, Sabrina Pagliarusco, Ellenia Bordini, Silvia Martinucci, and Mario Pellegatti Department of Drug Metabolism and Pharmacokinetics, Medicine Research Center, GlaxoSmithKline, Verona, Italy Received March 9, 2010; accepted July 9, 2010 ABSTRACT: Casopitant [1-piperidinecarboxamide,4-(4-acetyl-1-piperazinyl)-- ((1R)-1-(3,5-bis(trifluoromethyl)phenyl)-ethyl)-2-(4-fluoro-2-methylphenyl)--methyl-(2R,4S)] is a potent and selective antagonist of the neurokinin-1 (K1) receptor, developed for the prevention of chemotherapy-induced nausea and vomiting and postoperative nausea and vomiting. Absorption, distribution, metabolism, and elimination of [ 14 C]casopitant have been investigated in the mouse, rat, and dog after single oral administration and compared with those in humans. [ 14 C]Casopitant was rapidly absorbed in all three species: the maximum plasma concentration of radioactivity was generally observed 0.5 to 2 h after a single oral dose. In dog and female rat, as observed for humans, the principal circulating radiolabeled components were unchanged casopitant and its hydroxylated derivative M13. In rats, there was an evident sex-related Introduction eurokinin subtype 1 (K-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 and the dorsal motor nucleus of the vagus. They are activated by substance P (SP), a neuropeptide member of the tachykinin family. SP and its K-1 receptors are also found in non-neural tissues such as endothelial and inflammatory cells as well as in gastrointestinal, respiratory, and genitourinary tissues. K-1 receptor antagonists are believed to exert their antiemetic effect by blocking SP-mediated receptor activation within the central vomiting center, which is essential for the processing of emetic stimuli and the coordination of the emetic reflex (Sanger and Andrews, 2006; Diemunsch et al., 2009). Casopitant [1-piperidinecarboxamide,4-(4-acetyl-1-piperazinyl)-- ((1R)-1-(3,5-bis(trifluoromethyl)phenyl)-ethyl)-2-(4-fluoro-2- methylphenyl)--methyl-(2r,4s); GW679769], is a potent and selective antagonist of the human K-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 postoperative nausea and vomiting (Arpornwirat et al., 2006; Article, publication date, and citation information can be found at doi: /dmd difference in the rate of elimination of drug-related material with elimination being more rapid in males than in 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 - acetyl group, -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 for between 2 and 7% of the dose, with unchanged parent drug never quantifiable. Chung et al., 2006; Rolski et al., 2006; Singla et al., 2006; Aziz et al., 2008; Grunberg et al., 2008; Herrstedt et al., 2008; avari, 2008; Strausz et al., 2008). The metabolism and excretion of casopitant in humans have been reported recently (Pellegatti et al., 2009). In humans, 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. In this article, we describe the 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 [ 14 C]casopitant (ig. 1). Materials and Methods Chemicals. [ 14 C]Casopitant mesylate, [ 12 C]casopitant mesylate, standards of metabolites M12 (coded as GSK631832), M13 (coded as GSK525060), M31 (coded as GSK517142), M44 (coded as GSK ), and M134 (coded as GSK ), were all supplied by Chemical Development, GlaxoSmith- Kline. The structures of metabolites are reported (Table 2). Radiolabeled [ 14 C]casopitant was synthesized by GlaxoSmithKline Isotope Chemistry (Stevenage, UK); the specific activities were 0.37 MBq/mg for the mouse study, MBq/mg for the rat study, and MBq/mg for the dog study, and the radiochemical purity was at least 99%. Commercially obtained chemicals and solvents were of HPLC or analytical grade. Liquid scintillation Downloaded from dmd.aspetjournals.org at ASPET Journals on ovember 10, 2018 ABBREVIATIS: K-1, neurokinin-1; SP, substance P; GW679769, 1-piperidinecarboxamide,4-(4-acetyl-1-piperazinyl)--((1R)-1-(3,5-bis(trifluoromethyl)phenyl)-ethyl)-2-(4-fluoro-2-methylphenyl)--methyl-(2R,4S); SD, Sprague-Dawley; HPLC, high-performance liquid chromatography; BDC, bile duct-cannulated; LSC, liquid scintillation counting. 1876

2 METABLIC DISPSITI CASPITAT I PRECLIICAL SPECIES 1877 IG. 1. Structure of [ 14 C]casopitant. cocktails were obtained from Zinsser Analytics (rankfurt, Germany) and PerkinElmer Life and Analytical Sciences (Waltham, MA). Animals. Male and female SD rats [ g for intact rats and g for bile duct-cannulated (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 and Charles River (Lecco, Italy). The animals were kept under standard environmental conditions using routine methods of animal husbandry. Water from the domestic supply and a standard diet were provided to animals. The animals were not fasted overnight, but food was withheld at the time of dosing and was returned approximately 4 h after dosing. Water was available ad libitum. The rat study was conducted at Huntingdon Life Sciences (Cambridgeshire, UK); the dog and mouse studies at Charles River Laboratories and at Glaxo- SmithKline (Verona, Italy). All in-life experiments described in this article complied with national legislation and with the company policy on the care and use of laboratory animals and with related codes of practice. Dose Preparation and Sample Collection. The doses selected for rat and dog were the no observed adverse effect levels during 13- and 4-week toxicity studies, respectively, whereas for mouse it was the mean of the lowest doses used during toxicity studies up to 13 weeks of treatment. or the total mass balance and metabolism studies, oral doses were prepared by dissolving an appropriate amount of [ 14 C]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 analyzed by radio- HPLC before and after dosing to determine the radiochemical purity and confirm the stability of [ 14 C]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 [ 14 C]casopitant mesylate at a target dose level of 50 mg of free base/kg. Total radioactivity was determined in urine and feces collected up to 168 h after dosing. At the end of each excreta collection period, the cages were washed with water and acetonitrile as appropriate, and the wash was retained for radioassay. Radioactivity was also determined in the carcasses at 168 h after dosing. In addition, predose excreta were collected to provide control matrices and storage stability samples. Twenty-five male and 25 female mice were dosed with the same oral dose to investigate total radioactivity concentration in blood and plasma. ive animals per sex were bled at each time point. Blood collection was performed by heart puncture before dosing and at 0.5, 2, 6, and 24 h after dosing under isoflurane anesthesia. Blood samples were collected into tubes containing K3EDTA as the anticoagulant. After removal of aliquots for radioanalysis, the remaining blood samples were centrifuged at 2000g for 10 min at 4 C 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 [ 14 C]casopitant mesylate by gavage at 15 mg of free base/kg. Urine and feces were collected before dosing and over a 24-h period up to 168 h after dosing. At 168 h after dosing, 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 anesthesia (isoflurane) (van Wijk et al., 2001). ne end of a flexible cannula was inserted into the common bile duct of each rat, and the other end was inserted into the duodenum. The cannula loop was exteriorized, the incisions were closed, and a protective harness was fitted. The animals were allowed to regain consciousness. n the day before dosing, the cannula loop was opened. The BDC rats received a single oral dose of [ 14 C]casopitant at 15 mg of 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 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 before dosing and up to 96 h after dosing. Bile samples were collected overnight before dosing and during the periods 0 to 6 and 6 to 24 h and then over 24-h periods up to 96 h after dosing. At the end of each excreta collection period, the cages of both intact 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 anesthesia and then killed by cervical dislocation at time points of 2, 8, and 24 h after oral dosing (15 mg of free base/kg). Blood samples were collected, and plasma samples were 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 before dosing and over the period of 24 h up to 216 h. Blood samples were also collected (into tubes containing K3EDTA as anticoagulant) from the same animals at predose and at 1, 6, 24, and 96 h after a single oral dose of [ 14 C]casopitant mesylate at 10 mg of free base/kg. Plasma was separated by centrifugation as described previously. Urine and feces were also collected before dosing and at 24-h intervals up to 96 h from two additional male dogs that had been surgically prepared previously with indwelling bile duct cannulae, under general anesthesia (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, 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 approximately 2 h before dose administration to divert bile into the proximal cannula for collection via the access port (Kissinger et al., 1998). These dogs were administered the same dose as the previous ones. Bile samples were also collected from each BDC dog before dosing and over 0 to 6, 6 to 24, 24 to 48, 48 to 72, and 72 to 96 h after dosing. After dosing, the dogs were infused with replacement bile salt solution (18 g of cholic acid and 1.3 g of sodium bicarbonate in 1 liter of 0.9% w/v aqueous sodium chloride solution, ph adjusted to ) 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 was retained for radioassay. Assay of Total Radioactivity. Aliquots of urine, cage washes, and bile and plasma samples were mixed with either AquaSafe 500 plus, Ultima Gold, or Pico-luor 40M scintillator. eces samples were weighed, an appropriate amount of water was added, and the total weight was recorded before homogenization. Aliquots of each homogenate were combusted using a Packard Tri-Carb 307 Automatic Sample xidizer (PerkinElmer Life and Analytical Sciences). The resultant 14 C 2 was collected by absorption in Carbosorb (8 ml) to which PermafluorE scintillation fluid (10 ml) was added. Carcasses were solubilized in a caustic digestion solution containing aqueous sodium hydroxide, methanol, and Triton X-100. Aliquots of each digest were combusted as described above for fecal samples. Brain samples were homogenized with water (1:2, w/w) on ice. Aliquots of each brain homogenate and rat, mouse, and dog blood samples were combusted as described previously. inally, all samples were analyzed by using a Packard Tri-Carb 2100 TR liquid scintillation counter (PerkinElmer Life and Analytical Sciences), with Downloaded from dmd.aspetjournals.org at ASPET Journals on ovember 10, 2018

3 1878 MIRAGLIA ET AL. automatic quench correction by an external standard method (Botta et al., 1985). Preparation of Plasma and Brain Samples for Radio-Liquid Chromatography-Tandem Mass Spectrometry 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 approximately 3 volumes of organic solvent (methanol for mouse and dog and a mixture of 50:50 acetonitrile-methanol for rat). After centrifugation, the supernatants were removed, the pellets were resuspended in the same solvent and volume, and the process was 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 before radio-hplc analysis. Preparation of Excreta Samples for Radio-Liquid Chromatography- Tandem Mass Spectrometry Analysis. ecal homogenates and urine and bile samples (bile for rat and dog only) for each animal were obtained by pooling across sampling times by total weight ratio to generate sample pools containing 90% or greater of the radioactivity excreted in the matrix. or feces, sample pools were processed as described previously for plasma and brain samples. Weighed aliquots of each extract were radioassayed by LSC for the calculation of recovery after extraction and reconstitution before HPLC analysis. Urine and bile samples were centrifuged to remove any particulates and analyzed by LSC before and after centrifugation to determine whether any notable losses of radioactivity had occurred. Bile samples were diluted 1:5 with water before radio-hplc analysis. Quantification and Profiling of Metabolites. Because of the large number of chemically different metabolites, a variety of HPLC methods were used as described below. HPLC method 1 (used for analysis of rat and dog plasma, rat brain, and mouse, rat, and dog feces samples). The chromatographic instrument used consisted of an 1100 binary pump, autosampler, and column oven (50 C) (Agilent Technologies, Palo Alto, CA) using a Synergi Polar-RP column ( mm, 4 m; Phenomenex, Torrance, CA). The mobile phase consisted of 5 mm ammonium formate aqueous (ph 5 adjusted with formic acid) (solvent A) and 5 mm ammonium formate aqueous (ph 5) 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 min, followed by a linear increase to 100% by 65 min, with these conditions being maintained for a further 7 min. or female rat feces, after 60 min the gradient was followed by two linear increases: to 80% by 75 min and to 100% by 75.1, with these conditions being maintained for a further 10 min. 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), a 717 autosampler (Waters, Milford, MA), and a TCM (Waters) column oven (35 C) using a Synergi Hydro-RP column ( mm, 4 m; Phenomenex). 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 min, followed by a linear increase to 90% by 50.1 min, with these conditions being maintained for a further 5 min. HPLC method 3 (used for analysis of rat and dog bile samples). 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 min, followed by three further linear increases: to 43% B by 40 min, to 60% B by 65 min, and then to 100% B by 75 min. These conditions were maintained for a further 5 min. or the analysis of urine samples another HPLC method, not reported here, was used. 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. ull recoveries of 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 fraction collector model C LH200 (Intek- Services, Surrey, UK) into 96-deep well microtiter plates (LumaPlates) containing yttrium silicate solid scintillant (PerkinElmer Life and Analytical Sciences). Radioactivity was determined by scintillation counting (TopCount XT counter; PerkinElmer Life and Analytical Sciences). Radio-HPLC data from mouse feces samples only were captured on-line using a Packard lo-ne 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-T Ultima (Waters MS Technologies, Manchester, UK) tandem mass spectrometer (Morris et al., 1996). Electrospray ionization, in positive and/or negative mode, was used. The HPLC flow was split (1:5) between the mass spectrometer and a fraction collector (model C 204; Gilson Inc.), which was used for off-line radiodetection. Metabolites were identified on the basis of charged molecular ions, mass accuracy, and their collision-induced dissociation fragmentation (liveira and Watson, 2000). Authentic standards, when available, were used to compare chromatographic retention times and fragmentation patterns. or some metabolites, a definitive structure had been previously confirmed by 1 H MR (Plumb et al., 1999) in clinical studies (Pellegatti et al., 2009). ully characterized metabolites were designated by the letter M followed by a number; when a synthetic standard was available, a GSK code number was assigned. Results Plasma and Blood Radioactivity Concentrations in Mice, Rats, and Dogs. After a single oral administration of [ 14 C]casopitant to CD-1 mice (50 mg/kg), the highest levels of drug-related material in plasma were detected at 2hinmales and 0.5 h 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. After a single oral administration of [ 14 C]casopitant (15 mg/kg) to SD rats, maximum mean concentrations of radioactivity in blood and plasma were observed at 2- and 8-h after dosing in males and females, respectively. Concentrations in these matrices were greater in females Downloaded from dmd.aspetjournals.org at ASPET Journals on ovember 10, 2018 TABLE 1 Recovery of radioactivity in mice, rats, and dogs after a single oral administration of [ 14 C]casopitant Results are reported as mean (unless stated otherwise) S.D. Data are presented to one decimal place and are computer-generated and rounded appropriately. As a consequence, calculations of values yield minor deviations. Matrix Male: Intact (n 6) Mice (50 mg/kg) Rats (15 mg/kg) Dogs (10 mg/kg) emale: Intact (n 6) Male emale Male Intact (n 3) BDC (n 2) Intact (n 3) BDC (n 2) Intact (n 3) BDC (n 2) emale: Intact (n 2) Urine eces a 85.5 Bile.D..D..D D D a.d. Cage Wash Carcass D..D..D. Total b a 92.5.D., not determined. a Individual values have been reported because there were notable differences in the extent of elimination between the two animals. b The total collection period was 0 to 168 h for mice and rats (intact), 0 to 96 h for rats and dogs (BDC), and 0 to 216 h for dogs (intact).

4 METABLIC DISPSITI CASPITAT I PRECLIICAL SPECIES 1879 Underlined metabolites have been confirmed with either MR or comparison with standards. TABLE 2 Relevant metabolites of [ 14 C]casopitant Proposed Structure Positive M H or egative M H Ion MS Products Ions (m/z) 1 H MR (600 MHz, 1:1 AC-D 2 (Where Available) Casopitant H H H C 3 CH M H 489, 481, 346, 327, 298, 277, 241, 210, M H 601, 467, 318, 190, 167, 336, (brs, 1H), 7.59 (brs, 2H), 7.21 (dd, 1H), 6.88 (dd, 1H) 6.76 (dt, 1H), 5.32 (q, 1H, CH ), 4.23 (m, 1H), 3.49 (m, 1H), 2.86 (m, 1H), 2.69 (s, 3H, ), 2.34 (s, 3H), 1.46 (d, 3H, CH ), several piperidine and piperazine signals versus broad M1 M3 H M9 H tentative M10 H H GSK M H 615, 505, 481, 369, 362, 241, 210, 173, M H 489, 327, 298, 241, M H 489, 327, 298, 241, M H 489, 453, 249, 241, 182, (brs, 1H), 7.60 (brs, 2H), 7.25 (dd, 1H), 7.07 (dd, 1H) 6.84 (dt, 1H), 5.35 (q, 1H), 4.74 and 4.64 (d, 1H each, CH 2 H), 2.65 (s, 3H), 1.46 (d, 3H), several signals broad and/or obscured Downloaded from dmd.aspetjournals.org at ASPET Journals on ovember 10, 2018 H 633 M H 615, 497, 489, 479, 461, 275, 183, (brs, 1H), 7.59 (brs, 2H), 7.16 (m, 1H), 6.84 (dd, 1H) 6.72 (m, 1H), 5.81 and obscured (m, 1H, rotamers, CHH) 5.35, several signals broad and/or obscured GW H H 393 M H 336, 210, 208, 190, 167 M16 or isomer/ tautomer H 391 M H] 210, 190, 167 M17

5 1880 MIRAGLIA ET AL. TABLE 2 Continued Proposed Structure Positive M H or egative M H Ion MS Products Ions (m/z) 1 H MR (600 MHz, 1:1 AC-D 2 (Where Available) M21 H H 393 M H] MS/MS of M H] -H 2 : 344, 275, 239, 208, (dd, 1H), (m, 2H), 5.74 and 5.39 (m, 1H, rotamers, CHH), 2.49 (s, 3H), 2.34 (s, 3H) several signals broad and/or obscured HC H 391 M H] 192, 184, 167 M22 H 377 M H] 241, 210, (dd, 1H), (m, 2H), 2.49 (s, 3H), 2.34 (s, 3H) several signals broad and/or obscured M28 H H GSK (M31) M33 C H 3 M37 H H H H or isomers M41 CH3 H C 3 H H C H 3 GSK (M44) H 591 M H] 489, 455, 327, 320, 251, 241, 184, 175, M H] 617, 465, 291, 245, 208, 165, M H] 489, 467, 327, 298, 263, 241, 197, M H] 617, 505, 487, 336, 327, 298, 272, 241, 190, 173, M-H 415, 241, 213, M H] 332, 272, 241, 210, 204, (brs, 2H), 8.03 (brs, 1H), 5.18 (q, 1H), 4.09 (d, 1H, anomeric), 1.45 (d, 3H) Downloaded from dmd.aspetjournals.org at ASPET Journals on ovember 10, 2018 M50 than males at corresponding time points by up to 4-fold. In general, the circulating radioactivity was evenly distributed between the plasma and blood cellular fractions. In beagle dogs, after a single oral administration of [ 14 C]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 h after dosing. Brain Radioactivity Concentration in Rats. After a single oral dose of [ 14 C]casopitant (15 mg/kg) to SD rats, maximum mean

6 METABLIC DISPSITI CASPITAT I PRECLIICAL SPECIES 1881 Proposed Structure TABLE 2 Continued Positive M H or egative M H Ion MS Products Ions (m/z) 1 H MR (600 MHz, 1:1 AC-D 2 (Where Available) C H 3 H H H CH M H] 548, 489, 412, 327, 298, 241, 141 M57 H H 649 M H] 631, 465, 348, 330, 305, 222, 208, 165 M61 M63 M69 H H H H CH3 H C 3 H H H C 3 M70 M78 H H (H) M H] 467, 350, 332, 290, 222, 210, 204, M H] 475, 467, 346, 277, 241, 210, M H] 573, 308, 290, 208, 190, M H 523, 505, 487, 371, 327, 298, M H 645, 330, 305, 241, 208, 204 Downloaded from dmd.aspetjournals.org at ASPET Journals on ovember 10, 2018 M80 H H SG CH M H 767, 589, 571, 489, 453, 446, 308 M81, M82 concentrations of radioactivity in brain were observed at 2 and 8 h after dosing in males and in females, respectively. The 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 h after dosing. Total Mass Balance. Recovery of radioactivity in mice, rats, and dogs after a single oral administration of [ 14 C]casopitant is shown in Table 1. Excretion in mice. After a single oral dose of [ 14 C]casopitant to CD-1 mice at 50 mg/kg, there were no sex differences in the routes

7 1882 MIRAGLIA ET AL. TABLE 2 Continued Proposed Structure H M100, M102, M106, M118 C H 3 Positive M H or egative M H Ion MS Products Ions (m/z) 823 M H 805 (not for M118), 647, 519 (M100 only), 481 (not for M118), 332, 290 (not for M118), 272 (M100 only), 241(not for M118), 210, 204(not for M118), 167(not for M118) 809 M H] 633, 615, 505, 481, 362, 210, H MR (600 MHz, 1:1 AC-D 2 (Where Available) M103 M104 M109 M114 M115 M117 H H H H 3 S H H H H 683 M H] 507, 489, 397, 379, 327, 298, 241, M H] 489, 392, 327, 298, 241, M-H 269, 243, 223, 203, M-H 213, 193, M-H 257, 237, 213, M H] 807, 649, 631, 613, 505, 479, 467, 291, 245, 226, 208, 183, 165 Downloaded from dmd.aspetjournals.org at ASPET Journals on ovember 10, 2018 M119 H or H H H CH M H] 621, 184 M120

8 METABLIC DISPSITI CASPITAT I PRECLIICAL SPECIES 1883 TABLE 2 Continued Proposed Structure Positive M H or egative M H Ion MS Products Ions (m/z) 1 H MR (600 MHz, 1:1 AC-D 2 (Where Available) Unknown cysteine conjugate M122 H H 738 M H] 672, 489, 401, 327, 241, M H] 489, 428, 327, 315, 298, 272, 241, 224, 175, 157, 114 M123 H M H] 370, 315, 298, 241, 175 GSK (M134) M136 H H M137 M138 H H M141 or H H H H H H H () 2 H 449 M-H 273, 255, 243, 213, 193, M H] 304, 286, 276, 272, 241, 204, M H] MS/MS of M H] -H 2 ion: 344, 275, 239, 208, M H] 519, 455, 350, 306, 290, 241, 204, 184, M H] 489, 241, 200, 157 a: 7.92 b: 8.00 c: 5.04 d: 3.80,3.91. anomeric: (brs, 1H), 7.61 (brs, 2H), 7.26 (dd, 1H), 7.07 (dd, 1H) 6.84 (dt, 1H), 5.35 (q, 1H), 4.74 and 4.66 (d, 1H each, CH 2 H), 2.62 (s, 3H), several signals broad and/or obscured Downloaded from dmd.aspetjournals.org at ASPET Journals on ovember 10, 2018 M155 M169 H H H H M171, M172, M M H] 414, 327, 298, 279, 272, 241, 210, 184, 175, 143, M H] 821, 803 (not for M171), 663 (M171 only), 645 (M171 only), 479 (M173 only), 330 (not for M172), 305(M171 only), 272(M171 only), 208, 165 (M173 only) AC, acetonitrile; D 2, deuterium oxide.

9 1884 MIRAGLIA ET AL. and rates of elimination of radioactivity. The major route of elimination was via the feces (approximately 86% of the dose) with urinary excretion accounting for a mean of approximately 6%. Elimination of drug-related material was rapid in both sexes, with 85% of the administered dose recovered by 24 h. The mean total recoveries were 99%. TABLE 3 Percentage of radioactivity of casopitant and its relevant metabolites in mouse, rat, and dog plasma after a single-dose oral administration of [ 14 C]casopitant M44, M117 (male rat), M114, M115 (rat and dog), and M136 (dog) were nonradiolabeled metabolites observed by mass spectrometry only. bserved metabolite radioactivity was determined by 96-well fraction collection with scintillation counting for 5 min after a chromatographic separation was performed by HPLC. Percentages obtained from the radiochromatogram were adjusted for the extraction recovery. The percentage of radioactivity per time point does not equal 100% because only distinct radioactive peaks were assigned values and only those greater than 5% of plasma radioactivity in at a least one species were reported in the table. Plasma Radioactivity a Peak Mice (50 mg/kg), Male Male Rat (15 mg/kg) emale Dog (10 mg/kg), Male 0.5 h 6 h 2 h 8 h 2 h 8 h 1 h 6 h % Casopitant M ( M166) 4.9 ( M166) BQL.D. M9.D..D..D..D. 1.7 ( M112) 1.2 ( M112) M ( M69) 4.0 ( M69) M12 M69.D..D ( M154) 3.1 ( M154) M M D..D..D..D..D..D. M D..D..D..D..D..D. M D..D..D..D M31 M134.D..D M57 M155.D..D..D..D..D..D M109.D..D..D..D ( M1) BQL ( M1) Total g Eq/g in plasma BQL, below quantification limit, set to 25 cpm as peak area;.d., not detected. IG. 2. Representative reconstructed radiochromatograms of pooled male (a) mouse and (b) dog plasma after a single oral administration of [ 14 C]casopitant at a target dose of 50 and 10 mg of free base/kg, respectively. Downloaded from dmd.aspetjournals.org at ASPET Journals on ovember 10, 2018

10 METABLIC DISPSITI CASPITAT I PRECLIICAL SPECIES 1885 Excretion in rats. After a single oral administration of [ 14 C]casopitant (15 mg/kg) to intact SD rats, the major route of elimination of radioactivity was via the feces (a mean of 97% of the dose). Urinary elimination was minor (a mean of 3% of the dose). The mean total recovery of radioactivity was approximately 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 drug-related material found in the residual carcass. The mean total recovery of radioactivity was approximately 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, approximately 92% of the dose was recovered by 24 h after dosing in males compared with only 37% in females. Excretion in dogs. After a single oral administration of [ 14 C]casopitant to intact beagle dogs (10 mg/kg), the major route of elimination of radioactivity was via the feces (approximately 84% of the dose). Urinary excretion was a minor route of elimination (approximately 6% of the dose). Elimination of radioactivity was initially rapid with a mean of approximately 80% of the administered radioactivity recovered in the excreta and cage wash of either sex by 72 h after dosing. However, small amounts of radioactivity continued to be recovered, primarily in the feces, up to 216 h. The mean total recovery was approximately 91%. In BDC dogs, there were notable differences in the extent of elimination via the bile and feces among 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 interindividual variability in the biliary elimination is common in dogs, and no issues have been reported during the surgical procedure, it 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. 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 those of the other species: in male mice, the major metabolites were M28, resulting from cleavage of the molecule, and its oxidized derivatives and M16 and M17, ranging between 18 and 7% of plasma radioactivity at 0.5 h and between 6 and 1% at 6 h. emale 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 and female dog (female dog data not reported). The major IG. 3. Representative reconstructed radiochromatograms of pooled (a) male and (b) female rat plasma after a single oral administration of [ 14 C]casopitant at a target dose of 15 mg of free base/kg. Downloaded from dmd.aspetjournals.org at ASPET Journals on ovember 10, 2018

11 1886 MIRAGLIA ET AL. circulating metabolites resulted from different modifications of the piperazine ring including the hydroxylated derivative M13 and the -dealkylated piperazine derivatives 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. ther minor components observed in both species were the deacetylated and oxidized derivatives M9 and M12, the -demethylated M69, and two oxidized metabolites M10 and M109. Metabolites M57 and M155,,deethylated hydroxylated derivatives, were detected in the dog only. Quantification for some relevant metabolites in plasma after oral administration at selected time points is summarized in Table 3, and representative radiochromatogram profiles are shown in igs. 2 and 3. Brain. The principal radiolabeled component in male and female rat brain extracts was unchanged parent representing approximately 50 to 80% of the brain radioactivity. ther notable radiolabeled components were M13 (in male and female) and M3 (in male only), representing approximately 12 to 24 and 9% of the brain radioactivity, respectively. igure 4 shows representative radiochromatograms of male and female rat brain extracts. Quantitative results from brain samples are summarized in Table 4. eces. Radio-HPLC analysis of male and female rat, dog, and mouse fecal extracts after oral administration of [ 14 C]casopitant showed complex metabolic profiles, with many radiolabeled 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 5. Metabolite profiles of all IG. 4. Representative reconstructed radiochromatograms of pooled (a) male and (b) female brain rat after a single oral administration of [ 14 C]casopitant to Sprague-Dawley rats at a target dose of 15 mg of free base/kg. intact animals were qualitatively similar to those of the BDC animals; moreover, mouse and dog profiles were qualitatively similar between sexes (data not shown). Therefore, results from male intact animals only are presented, with the exception of rat, for which observed sex differences require some discussion on the female rat results too. In male mouse feces, the principal radiolabeled components were multiple-oxidized metabolites M50 and M80, representing together 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 [ 14 C]casopitant bserved metabolite radioactivity was determined by 96-well fraction collection with scintillation counting for 5 min after a chromatographic separation was performed by HPLC. Percentages obtained from the radiochromatogram were adjusted for the extraction recovery. The percentage of radioactivity per time point does not equal 100% because only distinct radioactive peaks were assigned values and a few minor metabolites were not reported. Peak Male Brain Radioactivity emale 2h 8h 2h 8h 24h % Casopitant M3 9.1 BQL.D..D..D. M12 M69 BQL BQL M BQL M31 a BQL 2.0.D. Total g Eq/g in brain BQL, below quantification limit, set to 25 cpm as peak area;.d., not detected. a, observed by HPLC-mass spectrometry only. Downloaded from dmd.aspetjournals.org at ASPET Journals on ovember 10, 2018

12 METABLIC DISPSITI CASPITAT I PRECLIICAL SPECIES 1887 TABLE 5 Mean percentage of administered dose of casopitant and its relevant metabolites in excreta after a single oral dose of [ 14 C]casopitant to mice, rats, and dogs M44, M114, M115, and M136 were nonradiolabeled metabolites observed in all tree species by mass spectrometry only. Percentages were obtained from the radiochromatogram 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, because not all the time points were pooled and a few minor metabolites were not reported. %Administered Dose a Peak Mice (50 mg/kg), Male Male Rat (15 mg/kg) emale Dog (10 mg/kg), Male eces (intact) Casopitant M1.D. 2.1 ( M41 M42) 6.8 ( M41) 2.5 ( M181) M3 1.1 ( M9 M205) M13.D. BQL M28 M D. M31 M ( M169) 1.3 ( M169) 9.0 ( M169) 3.3 M33.D. 1.0 ( M164) M D. M ( M137) ( M63).D. M ( M16) ( M61).D. M76.D. BQL 4.6 ( M111 M170).D. M ( M106) 1.1.D. M123.D. 1.2 ( M62 M70) 3.5 ( M62 M70) 4.6 Total % dose in feces Bile Casopitant.S. BQL M1 M42.S. 0.7 ( M41) M3.S. 0.4.D. 1.7 ( M9 M36) M12.S. BQL 0.5 ( M131) 1.2 M31 M134.S. BQL ( M13 M57) M33.S M37.S..D..D. 3.8 ( M81 M82) M50.S. 9.0 ( M81) 0.7.D. M63.S. 1.5 ( M78) 1.3.D. M70.S. 0.8 ( M62 M123) ( M123) M78.S. 1.5 ( M63) 1.5.D. M80.S. 3.7 ( M122 M174) 0.9 ( M81 M82).D. M100 M172.S. 6.9.D..D. M103.S. 9.6 ( M102 M106) 3.4 ( M104) 3.3 M104.S. 0.9 ( M79 M120) 3.4 ( M103) 1.9 ( M187) M118 M171.S. 2.9.D..D. M119.S. 5.2 ( M173) M120.S. 0.9 ( M79 M104) 1.7 ( M122).D. M122.S. 3.7 ( M80 M174) 1.7 ( M120).D. M123.S. 0.8 ( M62 M70) ( M70) M174.S. 3.7 ( M80 M122) 0.3.D. M185.S..D..D. 1.3 Total % dose in bile.s Urine (intact) Casopitant a BQL BQL M D. M BQL 0.5 ( M198) M ( M22) M D. 0.4 M Total % dose in urine Downloaded from dmd.aspetjournals.org at ASPET Journals on ovember 10, 2018.D., not detected; BQL, below quantification limit, set to 25 cpm as peak area;.s., no sample. a, observed by HPLC-mass spectrometry only. approximately 29% of the administered dose. ther minor metabolites were observed, including oxidized derivatives (M61, M63, and M137), cleavage metabolites such as M28 and M16 (oxidized M28 derivative), and -dealkylated piperazine oxidized derivative M141, each representing no more than 4 to 8% of the administered dose. The principal radiolabeled derivatives in intact male rat feces, as for the mouse, were metabolites M50 and M80, representing together approximately 60% of the administered dose. Instead, in female rat the fecal radioactivity was distributed among several radio-peaks, the major one of which included -dealkylated piperazine derivatives M31, M134, and M169, accounting together for approximately 9% of the administered dose and the coeluting metabolites M41 (oxidized derivative) and M1 (a -demethylated oxidized derivative), representing together approximately 7% of the administered dose. The remaining radio-components accounted individually in both sexes for less than 5% of the administered dose. igure 5a shows a representative radiochromatogram of female rat feces extract. The principal radiolabeled compounds in male dog feces were derived from methyl hydroxylation (M3) and either oxidation on the piperazine ring followed by -dealkylation (M31, M123, and M134) or -demethylation and further 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 [ 14 C]casopitant showed many radiolabeled components partially coeluting. Results are summarized in Table 5. emale and male rat bile radio-profiles were qualitatively similar, but individual radio-peaks differed in their relative abundance. In

13 1888 MIRAGLIA ET AL. male rat bile, several glucuronide oxidized derivatives were detected (M100, M102, M103, M106, M118, M119, M171, M172, and M173) accounting together for approximately 25% of the dose. Another notable radio-peak corresponded to coeluting metabolites M50 and M81 (-deacetylated hydroxylated glutathione conjugate), representing 9% of the dose. In female rat bile, each radio-peak accounted for 3% or less of the administered dose. The major radio-components observed were several oxidized glucuronide derivatives (M78, M103, M104, and M120) and a cysteine derivative (M122) together representing approximately 7% of the dose. -Demethylated oxidized derivatives (M1, M33, M63, and M70) together accounted for another 6% of the dose. igure 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% of the dose. Major metabolites observed included an oxidized derivative (M37), -deacetylated hydroxylated glutathione conjugates (M81 and M82) and two oxidized glucuronides (M103 and M119). In both rat and dog bile, unchanged parent was detected only in negligible quantities. Urine. Urine was a very minor route of excretion for all species; therefore, results 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. onradiolabeled metabolites. Metabolites derived from the cleavage of [ 14 C]casopitant with loss of the radiocarbon were also observed IG. 5. Representative radiochromatogram of (a) feces and (b) bile after a single oral administration of [ 14 C]casopitant to intact and bile duct-cannulated female Sprague-Dawley rats at a target dose level of 15 mg of free base/kg. 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 Tables 3 and 5. Discussion The absorption, metabolism, and elimination of [ 14 C]casopitant, after a 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 the maximum plasma concentration of radioactivity generally observed 0.5 to 2 h after single oral dosing. The routes of excretion of the drug-related material were similar in all preclinical species and also in humans (Pellegatti et al., 2009). Elimination of casopitant occurred almost exclusively by metabolism (with almost 200 metabolites identified across the different species and matrices) and mainly via 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 radioactivity was 90%, and for the most part radioactivity was eliminated within 96 h after dosing. Similar to humans, in the dog the rate of elimination of radioactivity was slow, with small amounts of radioactivity excreted up to 216 h. nly rats showed an evident sex-related difference in the rate of excretion of the drug-related material, with elimination in both intact Downloaded from dmd.aspetjournals.org at ASPET Journals on ovember 10, 2018

14 METABLIC DISPSITI CASPITAT I PRECLIICAL SPECIES 1889 and BDC animals being more rapid in males than females. Sexdependent differences in xenobiotic metabolism in rats can largely be attributed to differences in the profile of cytochrome 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 males (Mahnke et al., 1997). The 10 to 30% less total cytochrome P450 expression in females as opposed to that in males (Mugford and Kedderis, 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 transferases (Mugford and Kedderis, 1998), are sex-dependent, often showing higher activity in male than female rats. The 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 estrogens cause decreases in the biliary excretion of xenobiotics in rat (Vore and Montgomery, 1980). The hydrophilicity of these conjugate derivatives may favor their elimination compared with the less polar casopitant unconjugated metabolites of females, which tend rather to be retained and accumulated in the liver. ew conjugate derivatives were present in feces, suggesting that hydrolysis for most of metabolites occurred in the gastrointestinal tract. The casopitant metabolism observed in female rat and dog showed the greatest 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 with the dominant isoforms in female rat CYP3A9 and CYP3A62 (Matsubara et al., 2004) and canine CYP3A12 and CYP3A26. A summary of the observed metabolic pathways is reported in Table 6, and a simplified metabolic scheme based on the putative structure of metabolites is shown in ig. 6. The major human route of metabolism, involving modifications of the piperazine moiety, such as oxidations, ring opening and -dealkylation until loss of the ring, was the principal route in the dog and female rat also. 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 humans but were relevant in preclinical species, being observed primarily in rat and mouse. -Demethylation and -deacetylation were observed to different extents in humans and preclinical species, also in combination with extensive oxidation and conjugation, leading to very complex metabolic profiles. Cleavage of the molecule with loss of the radiocarbon and its further conjugation were 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 pathway of metabolism in all species. Because of the extensive number of metabolites detected, each one in the excreta generally accounted for only a 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 International Committee on Harmonization, 2009, human circulating metabolites after oral administration (Pellegatti et al., 2009), M13 and M12, were also the principal ones in these two species. They both resulted TABLE 6 Metabolic pathways observed in excreta of mice, rats, and dogs after single oral administration of [ 14 C]casopitant Percent dose figures are reported as a range because of metabolite coelutions. Glutathione Conjugation uronidation -Deacetylation and xidation -Dealkylation at Carbamate Group -Demethylation and xidation Methyl and Piperazine xidation Piperazine Ring pening and xidation Species (Gender) M81 Mice (male) M141 M3, M61, M50, M80 M63 M16, M20, M21, M28 M138 M137 %Dose A. Rat (male) M31, M123, M134, M169 M41, M50, M80 M1, M33, M63 M16, M20, M21, M28 M138 M100, M102, M103, M106, M118, M119, M171, M172 M173 %Dose Rat (female) M31, M123, M134, M169 M3, M13, M41, M50, M61,M80 M1, M33, M63 M20, M28, M138 M76 M78, M103, M104 M119 %Dose Dog (male) M31, M123, M134, M169 M3, M37 M1, M33 M16, M21 M28,M138 M9, M12 M103, M104, M119 M81, M82 %Dose A., not applicable, because mouse bile was not collected. Downloaded from dmd.aspetjournals.org at ASPET Journals on ovember 10, 2018