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)-(GW679769)] is a novel neurokinin-1 receptor antagonist being developed for the prevention of chemotherapy-induced and postoperative nausea and vomiting. The disposition of [14C]casopitant was determined in a single-sequence study in six healthy male subjects after single-dose 90-mg i.v. and 150-mg oral administration. Blood, urine, and feces were collected at frequent intervals after dosing. Plasma, urine, and fecal samples were analyzed by high-performance liquid chromatography/mass spectrometry coupled with off-line radiodetection for metabolite profiling. Moreover, urine was also analyzed with 1H-NMR to further characterize metabolites. Plasma pharmacokinetic parameters for casopitant, a major metabolite (M13, coded as GSK525060), and total radioactivity were determined. Absorption of radioactivity after oral administration appeared to be nearly complete; elimination was principally via the feces both after oral and intravenous administration. Urinary elimination accounted for only <8% of total radioactivity. The main circulating metabolites were a hydroxylated derivative, M13 (coded as GSK525060), and, after oral administration, a deacetylated and oxidized metabolite, M12 (coded as GSK631832). In addition, many other metabolites were identified in plasma and excreta: the principal route of metabolism included multiple oxidations, loss of the N-acetyl group, modifications or loss of the piperazine group, and cleavage of the molecule. Casopitant was extensively metabolized, and only negligible amounts were excreted as unchanged compound. Some phase II metabolites were also observed, particularly in urine.
Casopitant, also known as GW679769, is a novel, potent and selective orally available neurokinin-1 (NK-1) receptor antagonist, which has shown efficacy in the prevention of chemotherapy-induced nausea and vomiting (CINV) and postoperative nausea and vomiting (Arpornwirat et al., 2006; Chung et al., 2006; Rolski et al., 2006; Singla et al., 2006; Aziz et al., 2008; Grunberg et al., 2008; Herrstedt et al., 2008; Navari, 2008; Strausz et al., 2008). The site of action of NK-1 receptor antagonists for the prevention of emesis is believed to be the nucleus tractus solitarius, where vagal afferents from the gastrointestinal tract and inputs from the area postrema and other regions of the brain important in the control of emesis converge. For the prevention of nausea and vomiting, casopitant has been administered as a single dose or as part of an acute 3-day dosing regimen. Casopitant is administered in combination with a 5-HT3 receptor antagonist, such as ondansetron, and for the prevention of CINV, dexamethasone is also coadministered. Clinical studies have evaluated the safety and effectiveness of a single 50-mg oral dose of casopitant for postoperative nausea and vomiting and single (150 mg oral) or 3-day (90 mg i.v. or 150 mg oral on day 1, followed by 50 mg oral on days 2 and 3) regimens of casopitant for CINV.
The metabolism, excretion, and pharmacokinetics of casopitant in rats and dogs were studied (M. Pellegatti, P. Fizzotti, and E. Bordini, unpublished results) and showed that the compound is highly metabolized with metabolites eliminated mainly in the feces. The elimination half-life was 1.8 h in rats and 4.5 h in dogs. Metabolism and brain penetration have also been studied in the ferret (Minthorn et al., 2008).
The purpose of this study was to characterize the disposition and metabolism of [14C]casopitant (Fig. 1) after a single-dose intravenous and oral administration to healthy male subjects.
Materials and Methods
Chemicals. [14C]casopitant mesylate, casopitant, [13C,2H3]casopitant, standards of metabolites M13 (coded as GSK525060), M12 (coded as GSK631832), M31 (coded as GSK517142), M134 (coded as GSK2110277), M44 (coded as GSK1735004), and [13C,2H3]M13 were all supplied by Chemical Development, GlaxoSmithKline Research & Development (Verona, Italy and Stevenage, UK). The structures of metabolites are reported (see Table 4). Commercially obtained chemicals and solvents were of high-performance liquid chromatography (HPLC) or analytical grade. Liquid scintillation cocktails were obtained from Zinsser Analytics (Frankfurt, Germany) and PerkinElmer Life and Analytical Sciences (Waltham, MA).
Casopitant Formulations. Infusion solution was prepared in an infusion bag for intravenous administration by diluting [14C]casopitant at a concentration of 15 mg/ml (0.39 μCi/mg, radiochemical purity 100%) with sterile saline to reach a final concentration of 0.36 mg/ml in 250 ml (equivalent to 90 mg and 35 μCi). For oral administration, [14C]casopitant (0.5 μCi/mg, radiochemical purity 100%) was dissolved in sterile water and 10 ml were dispensed into individually labeled containers (equivalent to 150 mg and 75 μCi). The formulations were supplied to the study center by Pharmaceutical Development, GlaxoSmithKline (Ware, UK).
Study Design and Subjects. The clinical study was performed at Charles River Laboratories Clinical Services (Edinburgh, UK) in accordance with Good Clinical Practice and the principles of the 1996 version of the declaration of Helsinki. The protocol was reviewed and approved, as appropriate, by the Edinburgh Independent Ethics Committee for Medical Research and the Medicine and Healthcare products Regulatory Agency (MHRA). The proposed radioactive dose was approved by the Administration of Radioactive Substances Advisory Committee (ARSAC). Written consent was obtained from all subjects before any protocol-specific procedures.
Six healthy white male subjects, aged between 30 and 55 years and with a body mass index between 19 and 30 kg/m2, participated in this study. The study design was an open label, two-period single-sequence study with single-dose intravenous administration (infusion) followed by single-dose oral administration after a washout period. Subjects were in good health as assessed by clinical examination, nonsmokers, with no history of drug or alcohol abuse, and were on no other medication at the time of the study, with no prescribed medication within 14 days of the study commencing.
Study Procedures. Subjects were fasted overnight before casopitant administration. All subjects received a single intravenous infusion of [14C]casopitant (as mesylate salt) at a target dose of 90 mg (1.3 MBq, 35 μCi) in 250 ml of sterile saline over 15 min. After a washout period of 43 days, the same subjects received a single oral dose of 150 mg of [14C]casopitant (as mesylate salt) (2.8 MBq, 75μCi) dissolved in 10 ml of sterile water.
After intravenous administration, blood samples (7 ml for pharmacokinetic evaluation) were collected into K3-EDTA-containing tubes at predose, 0.25, 0.5, 1, 2, 3, 4, 6, 10, 16, 24, 36, 48, and 72 h after the start of infusion; additional larger samples for metabolite identification (30 ml) were collected at 0.25, 1, 3, and 6 h. After oral administration, blood samples (7 ml) were collected at predose, 0.5, 1, 2, 3, 4, 6, 10, 16, 24, 36, 48, and 72 h after casopitant administration; additional samples (30 ml) were collected at 1, 3, 6, and 16 h.
Blood samples were mixed, immediately chilled on crushed ice, and centrifuged for 10 min at 1500g at approximately 4°C to obtain plasma. Total radioactivity was measured on duplicate weighed aliquots of plasma; the remaining plasma was stored at –20°C before the assay for casopitant and M13 or profiling by HPLC. At selected time points (1, 3, 6, 16, and 24 h), blood radioactivity also was measured on duplicate aliquots of whole blood.
Urine samples were collected predose and between 0 to 6 h, 6 to 12 h, and 12 to 24 h after drug administration and then at 24-h intervals until 288 h. For each collection period, after thorough mixing, the weight of the sample was recorded, and radioactivity was determined on an aliquot of 5 ml; two 30-ml aliquots were stored at –20°C for metabolite identification.
Fecal samples were collected predose and at 24-h intervals until 288 h. Feces from each 24-h interval were mixed and homogenized 1:1 with water. Total weight of homogenate was recorded, and duplicate aliquots of 0.3 g were used for radioactivity determination; the remaining homogenate was stored at –20°C for metabolite identification.
Assay of Total Radioactivity. Aliquots of blood (250 μl) and of fecal homogenates (approximately 0.2 g) were combusted using a Packard model 307 sample oxidizer (PerkinElmer Life and Analytical Sciences). The 14CO2 generated was collected in a suitable absorbent scintillation system. Aliquots of liquid samples (e.g., urine and plasma) were subjected to liquid scintillation counting together with representative blank samples, the values of which were subtracted to give net dpm per sample, using a Liquid Scintillation Analyzer (PerkinElmer Life and Analytical Sciences) with automatic quench correction by an external standard method (Botta et al., 1985).
Casopitant and M13 Quantification. Quantification of casopitant and of M13 in plasma was performed by using a validated analytical method based on protein precipitation with acetonitrile, followed by HPLC tandem mass spectrometry (HPLC-MS/MS) analysis. Acetonitrile (150 μl) containing the internal standards ([13C,2H3]casopitant and [13C2,H3]M13, both at a concentration of 75 ng/ml) was added to plasma samples (50 μl). After vortex mixing, 100 μl of water was added and the deproteinized samples were centrifuged for 10 min at approximately 3000g. The supernatant was separated using a Betasil Phenyl Hexil column (50 × 3.0 mm, 5.0 μm; Thermo Fisher Scientific, Cheshire, UK) and eluted at a flow rate of 0.7 ml/min. The isocratic mobile phase consisted of 35% 5 mM ammonium acetate (native pH) and 65% acetonitrile. The column eluate was introduced into a TurboIonSpray source of a Sciex API-3000 triple quadrupole mass spectrometer (Applied Biosystems, Concord, Ontario, Canada) operated in positive-ion mode. The temperature of the probe was maintained at 350°C with a curtain gas setting of 10 and collision gas setting of 4. Casopitant and M13 were monitored by multiple reaction monitoring of 617→167 and 633→479, respectively. [13C,2H3]casopitant and [13C,2H3]M13 were monitored by multiple reaction monitoring of 621→171 and 637→483, respectively.
The concentrations of casopitant and M13 present in plasma samples were determined from calibration curves constructed from analysis of samples spiked with known concentrations of casopitant and M13, with weighted 1/x2 linear regression applied in each case over the range 1.5 to 1500 ng/ml.
Quality control (QC) samples, prepared at three different analyte concentrations and stored with study samples, were analyzed with each batch of samples against separately prepared calibration standards. QC samples and calibration standards were prepared using independently prepared stock solutions of casopitant and M13 reference materials. For the analysis to be acceptable, no more than one third of the QC results were to deviate from the nominal concentration by more than 15%, and at least 50% of the results from each QC concentration were to be within 15% of nominal.
Quantification and Profiling of Metabolites in Plasma, Urine, and Feces. Plasma samples from each time point were analyzed separately for each subject. Representative fecal and urine samples for each subject were obtained by pooling across sampling times by total weight ratio to generate sample pools containing 90% or greater of the radioactivity excreted in feces or urine.
Plasma samples (∼10 ml), after thawing, were centrifuged and diluted 1:1 v/v with 10% formic acid before being extracted by solid-phase extraction (SPE). Each sample was equally split and loaded onto two Oasis MCX (6 ml/500 mg) cartridges (Waters, Milford, MA), previously conditioned by rinsing with 5 ml of methanol and then 5 ml of water. Each column was washed with 3 ml of water, and radioactivity was eluted with 7 ml of ammonium hydroxide (5%) in methanol. The two extracted samples were combined, evaporated, and reconstituted in 1 ml of 80:20 water/methanol.
Urine samples (40 ml) were centrifuged and diluted 4:1 v/v with 10% formic acid before being extracted by SPE. The urine mixture was applied onto Oasis MCX (20 ml/1g) cartridges, previously conditioned with 15 ml of methanol and 10 ml of water. Cartridges were then washed with 5 ml of water, and the radioactivity was eluted with 9 ml of ammonium hydroxide (5%) in methanol. The extracts were evaporated and reconstituted in 1 ml of 80:20 water/methanol.
Fecal homogenates (approximately 4 g) were extracted by mixing with 7 ml of methanol/acetonitrile (50:50). After centrifugation, the pellet was extracted with 9 ml of methanol, centrifuged again, and extracted a third time with 9 ml of methanol. Supernatants were combined and concentrated under nitrogen to 1 ml before HPLC analysis.
Weighed aliquots of each extract were radioassayed by liquid scintillation counting for the calculation of recovery after extraction and reconstitution.
Because of the large number of metabolites and the qualitative differences across the various matrices, two different HPLC methods were used as detailed below.
HPLC method 1 (used for analysis of plasma and feces samples). The chromatographic instrument used consisted of an Agilent 1100 binary pump, autosampler, and column oven (50°C) (Agilent Technologies, Santa Clara, CA) using a Phenomenex (Torrance, CA) Synergi Polar RP column (250 × 4.6, 4 μm). The mobile phase consisted of 5 mM ammonium formate (pH 5) (solvent A) and 5 mM ammonium formate (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 an additional 5 min.
HPLC method 2 (used for analysis of human urine samples). The chromatographic method is as listed for method 1, with the exception of a different gradient, starting at 5% of B with a linear increase to 20% over 15 min, followed by four additional linear increases: to 25% B by 40 min, to 45% B by 45 min, to 80% B by 65 min, and then to 100% B by 65.1 min. These conditions were maintained for an additional 5 min.
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 the injected radioactivity. Full recoveries of radioactivity were obtained from the HPLC eluate collected.
Radio-HPLC data were captured off line (Bruin et al., 2006): chromatographic fractions were collected using a PerkinElmer fraction collector Intek-Services FC LH200 onto 96-deepwell LumaPlates microtiter plates containing yttrium silicate solid scintillant (PerkinElmer Life and Analytical Sciences). Radioactivity determination was performed by scintillation counting (Top-Count NXT counter; PerkinElmer Life and Analytical Sciences).
For NMR analysis of urine samples, 150 ml of urine were freeze-dried and reconstituted with 90:10 water/methanol (20 ml). After centrifugation, the sample was fractioned by preparative HPLC, using a chromatographic method as listed for method 2 but with a flow rate of 4 ml/min and a column dimension of 250 × 10 mm.
Structural Identification of Metabolites. Structural characterization was performed on selected samples by HPLC-mass spectrometry using hybrid quadrupole/time-of-flight Q-TOF Ultima (Waters MS Technologies, Manchester, UK) tandem mass spectrometer (Morris et al., 1996). Electrospray ionization, in positive or negative mode, was used. The HPLC flow was split (1:10) between mass spectrometer and a fraction collector model FC 204 (Gilson Inc., Middleton, WI) used for off-line radiodetection.
Metabolites were identified based on charged molecular ions, mass accuracy, and their collision-induced dissociation fragmentation (Oliveira and Watson, 2000). Authentic standards, when available, were used to compare chromatographic retention times and fragmentation patterns. Supporting data from preclinical studies (M. Pellegatti, P. Fizzotti, and E. Bordini, unpublished data) were also used in the assignment of metabolite structures. For some metabolites, confirmation of the structure has been obtained by 1H-NMR (Plumb et al., 1999).
NMR experiments were performed using a Bruker DRX-600 spectrometer (Bruker, Rheinstetten, Germany) equipped with an inverse 5-mm TXI Cryo-Probe (Bruker) (1H/13C/15N) operating at 600.13 MHz under the control of TopSpin. 1H-NMR spectra were acquired using a standard NOESYPRESAT pulse sequence for solvent suppression with time-shared double presaturation of the water and acetonitrile frequencies. In these experiments, 256 transients were acquired into 64 K data points over a spectral width of 12019 Hz (20 ppm) with an interscan delay of 2.4 s giving a pulse repetition time of 5 s. Fully characterized metabolites were designated by the letter M followed by a number; where a synthetic standard was available, a GSK code number was assigned.
Pharmacokinetic Analysis. Actual blood collection times and the actual dose administered were used for all pharmacokinetic calculations. Standard noncompartmental methods were used to derive pharmacokinetic parameters using WinNonlin (version 4.1; Pharsight, Mountain View, CA). Maximum plasma concentration (Cmax) and time of Cmax (tmax) were taken directly from the pharmacokinetic concentration-time data. Area under the plasma concentration-time curve (AUC) was calculated using the linear up-log down method, to the last sampling time t [AUC(0-t)], and extrapolated to infinity [AUC(0-∞)] by the addition of the concentration at the last sampling time divided by the terminal elimination rate constant, λz. Half-life was determined as ln(2) divided by λz. After intravenous administration only, systemic clearance (CL) was calculated as dose divided by AUC(0-∞), and the volume of distribution at steady state (Vdss) was the product of CL and the mean residence time as determined by moment analysis. The ratio of oral-to-intravenous dose-normalized AUC(0-∞) parameters were used for determination of the absolute bioavailability of the casopitant oral solution. Concentration units for radioactivity are expressed as nanogram-equivalents of casopitant/milliliter (ng-eq/ml).
Results
Mass Balance of Total Radioactivity. The actual dose of intravenous and oral casopitant administered ranged from 68 to 76 mg (27–30 μCi) and 141 to 144 mg (70–72 μCi), respectively. Subjects received a slightly lower dose of intravenous casopitant than was planned, most likely a result of incomplete flushing of the injection port of the intravenous infusion bag during preparation. This fact did not affect the aims of the study. The cumulative excretion of total radioactivity after single-dose intravenous and oral administration of [14C]casopitant to healthy male subjects at a target dose of 90 mg and 150 mg, respectively, is shown in Table 1.
After intravenous administration of [14C]casopitant in period 1, total radioactivity was eliminated primarily in the feces, accounting for a mean of 83.8% (range from 74.1 to 91.1%) of the administered dose by 288 h postdose. An additional fecal sample was collected from all subjects (408 h and/or 432 h postdose), and radioactivity in these samples ranged from 0.2 to 1.4% of the administered dose.
After a 43-day washout, following oral administration of [14C]casopitant in period 2, total radioactivity was eliminated primarily in the feces, accounting for a mean of 71.4% (range from 66.3 to 82.0%) of the administered dose by the end of the collection period. At 288 h postdose, radioactivity was still detectable in fecal samples (0.8–2.8%). By comparing the amount of total radioactivity in the urine from intravenous and oral administration of [14C]casopitant, and assuming linear pharmacokinetics and no enterohepatic recirculation, the mean fraction of casopitant absorbed after oral solution administration was estimated to be 93%.
Pharmacokinetics. A summary of the plasma pharmacokinetic parameters for casopitant, the major circulating metabolite M13, and total radioactivity after intravenous and oral administration is presented in Table 2. Median plasma concentrations of casopitant, M13, and total radioactivity (plasma and whole blood) after intravenous and oral administration are shown in Fig. 2. After oral administration, absorption of casopitant solution was rapid with a median tmax of 1 h, and the mean absolute bioavailability of casopitant oral solution was determined to be 0.57 (95% confidence interval: 0.39, 0.84). After intravenous and oral administration, the metabolite M13 was rapidly formed, and the terminal half-life of casopitant and M13 were similar. The AUC of M13 (based on molar concentrations) was 39 to 41% and 81 to 83% that of casopitant after intravenous and oral administration, respectively. The apparent terminal half-life of total radioactivity in plasma ranged from 69.4 to 172 h after intravenous administration and 68.6 h to 91.7 h after oral administration. Predose plasma samples collected before oral administration of [14C]casopitant (period 2) still contained levels of radioactivity slightly higher than background, indicating that a small amount of circulating radioactivity had remained after the intravenous dose, consistent with the long half-life of plasma radioactivity.
After both intravenous and oral administration of [14C]casopitant, mean blood/plasma total radioactivity ratios ranged from 0.64 to 0.71 over the first 24 h after intravenous administration and from 0.47 to 0.76 over the first 24 h after oral administration. Given the mean hematocrit of 0.4 observed in this study, the percentage of radioactivity associated with the cellular fraction of whole blood over the first 24 h postdose was 6 to 15% after intravenous administration and 0 to 21% after oral administration of casopitant.
Metabolic Profile.Plasma. The mean recovery of radioactive material after SPE of human plasma samples decreased at later time points after both intravenous and oral administration, from approximately 99 to 84% (i.v.) and from approximately 90% to approximately 65% (oral). Figure 3 shows two representative radiochromatogram profiles at 1 h after intravenous and oral dose. Quantification for some relevant metabolites after intravenous infusion and oral administration at selected time points are summarized in Table 3.
After intravenous administration, the percentage of radioactivity accounting for parent compound decreased from 92% at 0.25 h to 39% at 6 h. The only notable circulating metabolite was M13; there were several other circulating metabolites each accounting for less than 2% of plasma radioactivity. After oral administration, casopitant represented the major circulating component up to 16 h, decreasing on average from approximately 45% at 1 h to approximately 14% at 16 h. The major metabolites observed were M13 and M12. Other relevant metabolites were M31, M134, M2, M3, M10, M39, and M156. Some pooled plasma samples beyond 16 h were also analyzed (up to 72 h postdose): at these later time points, some oxygenated derivatives of M39 were prevalent, particularly M156. M44, a nonradioactive glucuronide of a cleavage derivative, was identified and estimated by HPLC-MS/MS using a biological standard (data not reported): this metabolite was less than 5 and 10% of the parent compound in intravenous and oral plasma extracts, respectively. Proposed structures and supporting spectral data are shown in Table 4.
Urine.Figure 4a shows a representative radiochromatogram of urine extracts. Urinary profiles were similar after intravenous and oral administration. Nonradioactive metabolites, resulting from cleavage, represented most of the drug-related material in the urine and were identified by 1H-NMR as well as HPLC-MS/MS. The main component was the glucuronide M44, estimated by 1H-NMR to account for 65% of the drug-related material in the urine. Other cleavage products identified were the N-dealkylated metabolite M28 and its hydroxylated derivatives M21, M138, and M139. Parent casopitant was present only in negligible amounts (<0.1% of the dose). Other metabolites were identified, but each accounted for less than 0.2% of the dose. Proposed structures and supporting spectral data are shown in Table 4.
Feces. Radio-HPLC analysis of fecal extracts after both intravenous and oral administration of [14C]casopitant showed many radiocomponents. Metabolic profiles were similar after oral and intravenous administrations. A representative radiochromatogram of fecal extracts is shown in Fig. 4b.
Because higher amounts of radioactivity were administered by the oral route compared with the intravenous route, the summary below is restricted to the fecal metabolite profile after oral administration, where enhanced radiochemical signals facilitated metabolite identification and quantification.
More than 40 metabolites were identified in feces, but the majority did not represent more than 1% of the dose. The principal radiolabeled compounds in feces were derived from oxidation and opening of the piperazine ring, together with multiple oxidations of the parent structure. Among these, M123 and M137 accounted for an average of 5% of the fecal radioactivity each (approximately 3.5% of the dose). M123 was derived from loss of the N-acetyl group and extensive metabolism of the piperazine ring to most likely an acid moiety; M137 lost the N-acetyl group and had both the piperazine and the fluorobenzylmethyl group oxidized. N,N-Deethylated-oxidized derivative M141 accounted for approximately 4% of the fecal radioactivity (approximately 3% of the dose).
Several other phase I metabolites (e.g., M50, M63, M73, M77, M80, M140, M146, and M157) each represented 2 to 3% of radioactivity in the feces, corresponding to less than 2% of the dose. Proposed structures and supporting spectral data are shown in Table 4. Parent was detected only in negligible quantities.
Discussion
Single-dose [14C]casopitant was administered in a single-sequence study to six healthy male subjects by both the intravenous and oral routes. Casopitant was rapidly and almost completely (estimated to be 93%) absorbed after oral solution administration, and the absolute bioavailability was determined to be 57%, suggestive of presystemic metabolism. After intravenous and oral administration, total radioactivity was eliminated primarily in feces, with urinary excretion of total radioactivity totaling less than 8% of the dose after the 9-day collection period. Total radioactivity was eliminated slowly and still detectable in fecal samples at the end of the continuous collection period (12 days).
The slightly lower total recovery after oral administration is most likely a result of small amounts of radioactive species that are slowly eliminated and confounded by the technical difficulties in the accurate determination of small amounts of radioactivity in feces samples at later time points. This result is not an uncommon finding with highly metabolized compounds (Roffey et al., 2007).
Systemic exposures to total radioactivity in plasma [AUC(0-t) and AUC(0-∞), respectively] were approximately 5.63- and 14.5-fold (i.v.) and 13.1- and 27.6-fold (oral) greater than corresponding estimates for the parent compound casopitant, demonstrating more extensive metabolism after oral administration. These data are also consistent with prehepatic and presystemic metabolism of casopitant. In addition, mean half-life estimates were appreciably longer for total radioactivity compared with parent casopitant. This observation, together with greater systemic exposure observed for total radioactivity, cannot be accounted for by casopitant and the major metabolite M13 alone, equals it reflects the formation of other radiolabeled metabolites or breakdown products that persist in the circulation longer than the parent compound.
Systemic plasma exposures to major metabolite M13 (based on molar AUC ratios) were between 39 to 41% (i.v.) and 81 to 83% (oral) of the corresponding estimates for parent casopitant. M13 and other metabolites (associated with total radioactivity) after both oral and intravenous administration appear rapidly, and the similar half-life estimates for casopitant and M13 also suggest that this metabolite is formation-rate limited, whereas some other metabolites (associated with total radioactivity) may be elimination-rate limited.
Mean estimates of casopitant systemic plasma clearance were low compared with both normal hepatic and renal plasma flow rates in man, suggesting that clearance of casopitant from plasma would not be limited by perfusion rates, and the large estimates of volume of distribution at steady state were appreciably greater than total body water and indicate that casopitant distributes extensively (Davies and Morris, 1993).
The identification of many metabolites in complex matrices such as plasma, urine, and feces posed a significant analytical challenge; HPLC-MS/MS in combination with off-line radioactivity monitoring, and in some instances 1H-NMR, were used for metabolite identification. For metabolite quantification, HPLC separation followed by off-line radioactivity measurement was used to increase sensitivity.
Based on the structure of metabolites, a putative simplified metabolic scheme is shown in Fig. 5. After oral administration, the principal route of metabolism involved multiple oxidations of the parent molecule, loss of the N-acetyl group, oxidation with progressive opening, and modification of both the piperazine and piperidine rings. N-Demethylation was also observed. A relevant route of metabolism observed in urine was the cleavage of the molecule with formation of some radiolabeled phase I metabolites (M28 and its hydroxylated derivatives) and of nonradiolabeled phase II metabolites, mainly M44.
The principal circulating metabolites after single oral administration were M13 and M12. Their affinity to NK-1 receptors was determined in vitro and was similar to casopitant (data not presented). However, because these metabolites have reduced brain penetration when compared with casopitant in animals (Minthorn et al., 2008), and lower clinical plasma exposure than casopitant (Tables 2 and 3), their contribution to the overall pharmacological activity of casopitant is expected to be small.
During the clinical development of casopitant, validated assays for M13 and M12 were established and demonstrated that these metabolites were present in the plasma of animal species used for toxicological investigations at concentrations similar to or higher than those determined in humans; therefore, their contribution to the overall toxicity of casopitant was established during standard toxicological investigations.
Both casopitant and M13 are primarily metabolized by CYP3A4 and show some potential for inhibition of CYP3A4 in vitro (unpublished results). A series of clinical drug-drug interaction studies have been conducted (Adams et al., 2009; Johnson et al., 2009), and further results will be the subject of future publications.
In conclusion, casopitant is well absorbed after oral administration and undergoes first-pass metabolism, resulting in an absolute bioavailability of approximately 60%. After both oral and intravenous administration, casopitant is extensively metabolized; metabolites are excreted primarily in feces. Two major circulating metabolites were characterized, M13 and M12, and whereas many other metabolites were identified, none represented more than 5% of the exposure to drug-related material.
Acknowledgments
We thank Ken Lawrie for synthesizing the labeled compounds, Mirka Scarati and Paul Cummings for the preparation of formulations, Heather Charles and Dixon Janet from Charles River, Roberta Mastropasqua for HPLC/MS quantification assays, Sabrina Pagliarusco and Silvia Martinucci for coordination of activities and helpful discussion, and the entire casopitant Clinical Pharmacology team for the design, conduct, and analysis of the clinical portions of the study.
Footnotes
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.109.026781.
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ABBREVIATIONS: 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)-; NK-1, neurokinin-1; CINV, chemotherapy-induced nausea and vomiting; HPLC, high-performance liquid chromatography; HPLC-MS/MS, HPLC tandem mass spectrometry; QC, quality control; SPE, solid-phase extraction; AUC, area under the plasma concentration-time curve; Cmax, maximal plasma concentration; tmax, time to reach Cmax; CL, systemic plasma clearance; Vdss, steady-state volume of distribution.
- Accepted May 4, 2009.
- Received January 27, 2009.
- The American Society for Pharmacology and Experimental Therapeutics