Metabolism and Excretion of the Novel Antipsychotic Drug Ziprasidone in Rats after Oral Administration of a Mixture of C- and H-labeled Ziprasidone

The metabolism and excretion of ziprasidone (5-[2-{4-(1,2benzisothiazol-3-yl)piperazin-1-yl}ethyl]-6-chloroindolin-2-one hydrochloride hydrate) were studied in Long Evans rats after oral administration of a single dose of a mixture of Cand H-labeled ziprasidone. The radioactive dose was quantitatively recovered over 7 days in both male and female rats. The percentage of the dose excreted in urine, bile, and feces of rats was 21.6, 19.2, and 55.6%, respectively. The total excretion in urine and bile suggested that at least 41% of the drug was absorbed. Absorption of ziprasidone was rapid, and the mean plasma concentrations of the unchanged drug and metabolites were slightly higher in the female rats than in the males. The maximal plasma concentrations for ziprasidone and metabolites were reached at 1 hr in both male and female rats. Based on AUC (0–12 hr) values, approximately 59 and 52% of the circulating radioactivity (average of C and H) was attributable to metabolites in male and female rats, respectively. Ziprasidone was extensively metabolized in rats, and only a small amount of ziprasidone was excreted as unchanged drug. Twelve metabolites were identified by ion spray LC/MS, using a combination of parent ion and product ion scanning techniques. The structures of eight metabolites were unambiguously confirmed by coelution on HPLC with synthetic standards, and four additional metabolites were partially identified. There was a gender-related difference in the excretion of urinary metabolites in Long Evans rats. The major route of metabolism in male rats involved N-dealkylation. In female rats the major metabolites were due to oxidation at the benzisothiazole ring. Based on the structures of these metabolites, four major and two minor routes of metabolism of ziprasidone were identified. The major routes included 1) N-dealkylation of the ethyl side chain attached to the piperazinyl nitrogen, 2) oxidation at the sulfur, resulting in the formation of sulfoxide and sulfone, 3) oxidation on the benzisothiazole moiety (other than sulfur), and 4) hydration of the CAN bond and subsequent oxidation at the sulfur of the benzisothiazole moiety. The minor routes involved N-oxidation on the piperazine ring and hydrolysis of the oxindole moiety. Classical antipsychotic drugs of the phenothiazine and butyrophenone classes have been established as effective agents for the treatment of schizophrenia, a mental disorder estimated to afflict 1% of the world population. However, these drugs are not effective in all patients or against all symptoms (1, 2). Secondly, an unacceptably high incidence of extrapyramidal symptoms limits the usefulness of these drugs (3). Laboratory and clinical findings have suggested that antagonism of serotonin 5-HT2 1 receptors in the brain limits the undesirable motor side effects associated with dopamine receptor blockade and improves efficacy against the negative symptoms of schizophrenia (4–9). ZIP is the hydrochloride salt of a benzisothiazolylpiperazine analog structurally related to the atypical antipsychotic drug tiospirone (10, 11). It was developed during a structure-activity investigation to find a compound that potently blocks dopamine D2 receptors while binding with even greater affinity to cerebral serotonin 5-HT2 receptors (12–14). ZIP has extremely potent central serotonin 5-HT2 (Ki 5 0.42 nM) and potent dopamine D2 (Ki 5 4.8 nM) receptor antagonistic properties. The ratio between these two affinities (5-HT2:D2 5 11.4) is greater than observed for other antipsychotic drugs (12–14). In phase II clinical studies, ZIP has demonstrated good tolerability, particularly with regard to extrapyramidal symptoms, at doses that are associated with efficacy in patients with schizophrenia (15). The oral bioavailability of ZIP in human is 59% and its elimination half-life is 4 hr (16). ZIP is well tolerated in animals at doses producing effective blockade of dopaminergic behaviors and has a favorable separation between activities predictive of efficacy and side effect liability. ZIP is partially absorbed in rats and dogs, with absolute oral bioavailability of 39–60%. The elimination half-life is about 1 hr in rats and 2.3 hr in male dogs (17). As with all new drugs, it is necessary that the metabolic fate of ZIP be evaluated in vivo. Metabolic studies conducted in living animals provide the ultimate information regarding metabolic pathways and disposition of the molecule that can be used to correlate or interpret efficacy and toxicity data. In addition, the identified metabolites can be evaluated for pharmacological activity. The objective of the present study was to investigate the metabolism and excretion of ZIP in LE This work was presented in part at the 6th North American International Society for the Study of Xenobiotics Meeting, Raleigh, NC, 1994. 1 Abbreviations used are: 5-HT, 5-hydroxytryptamine; ZIP, ziprasidone or CP88,059–01 (5-[2-{4-(1,2-benzisothiazol-3-yl)piperazin-1-yl}ethyl]-6-chloro-1,3-dihydroindol-2-one hydrochloride hydrate); LE, Long Evans; b-RAM, radioactivity monitor; ZIP-SO, ziprasidone sulfoxide; ZIP-SO2, ziprasidone sulfone; OH-ZIP, 5-hydroxyziprasidone; ZIP-NO, 5-[2-{4-(1,2-benzisothiazol-3-yl)-1-oxypiperazin1-yl}ethyl]-6-chloro-1,3-dihydroindol-2-one; BIT, 1,2-benzisothiazole; BITP, 3-(piperazin-1-yl)-1,2-benzisothiazole; BITP-SO, 3-(piperazin-1-yl)-1,2-benzisothiazole sulfoxide; BITP-SO2, 3-(piperazin-1-yl)-1,2-benzisothiazole sulfone; OX-Cl, 6-chloro-5-(2-chloroethyl)-1,3-dihydroindol-2-one; FAB, fast atom bombardment; CID, collision-induced dissociation. Send reprint requests to: Chandra Prakash, Ph.D., Department of Drug Metabolism, Central Research Division, Pfizer Inc., Groton, CT 06340. 0090-9556/97/2501-0206–218$02.00/0 DRUG METABOLISM AND DISPOSITION Vol. 25, No. 1 Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. 206 at A PE T Jornals on Jne 2, 2017 dm d.aspurnals.org D ow nladed from rats after administration of an oral dose of a mixture of Hand C-labeled ZIP (fig. 1). The use of two labels greatly facilitated the tracing and identification of metabolites formed through cleavage of ZIP, as observed for the structurally related drug tiospirone (18, 19). Materials and Methods General Chemicals. Commercially obtained chemicals and solvents were of HPLC or analytical grade. b-Glucuronidase (from Helix pomatia, type H-1, with sulfatase activity) was obtained from Sigma Chemical Co. (St. Louis, MO). YMC basic columns were obtained from YMC (Wilmington, NC). Ecolite-(1) scintillation cocktail was obtained from ICN (Irvine, CA). Carbsorb and Permafluor scintillation cocktails were purchased from Packerd Instrument Co. (Downers Grove, IL). Diazomethane was generated just before use from N-methyl-N-nitroso-p-toluene sulfonamide obtained from Aldrich (Milwaukee, WI). Silica gel was obtained from Fisher Scientific (Springfield, NJ). General Instrumentation. The structures of all synthetic compounds were supported by their H-NMR (Bruker AM-300) and FAB mass spectra. Melting points were determined on a Thomas-Hoover capillary melting point apparatus and are uncorrected. FAB mass spectra of synthetic standards were obtained on a VG-70/70 mass spectrometer, using glycerol as a matrix. Radiolabeled Drug and Reference Compounds. [C]ZIP, specifically labeled at C2 of the ethyl group attached to the piperazinyl nitrogen, was synthesized by Friedel-Crafts acylation of 6-chlorooxindole with [C]chloroacetyl chloride, followed by triethylsilane reduction of the aryl carbonyl and coupling with BITP (20). [C]ZIP showed a specific activity of 9.0 mCi/mmol (21.8 mCi/mg) and a radiochemical purity of $98%, as determined by radioHPLC. [H]ZIP, specifically labeled at the C7-position of the benzisothiazole ring, was synthesized by reduction of 7-bromo-ZIP with tritium gas (20). [H]ZIP showed a specific activity of 20.66 Ci/mmol (50.0 mCi/mg) and a radiochemical purity of $99% (by radio-HPLC). BITP, BITP-SO, BITP-SO2, BITP-SO2-lactam, OX-Cl, and OH-ZIP were prepared as described previously (20, 21). ZIP-N-oxide was prepared by reaction of ZIP with m-chloroperbenzoic acid. ZIP-SO. A mixture of BITP-SO (75 mg, 0.32 mmol), OX-Cl (73 mg, 0.32 mmol), Na2CO3 (100 mg, 0.96 mmol), and KI (5 mg, 0.03 mmol) in 1.0 ml of acetonitrile was stirred and heated to reflux for 32 hr. After cooling, the solvent was removed in vacuo and the residue was redissolved in 5 ml of anhydrous CH3OH, filtered through a pad of diatomaceous earth, and concentrated in vacuo to a brown oil. Chromatography on silica gel (40 mm) eluted with CH2Cl2/CH3OH (96:4) gave the desired product as a pale yellow solid [20 mg; m.p. 215–218°C; FAB mass spectrum: m/z 431 (Cl), 429 (MH); H-NMR (dimethylsulfoxide-d6): d 10.45 (s, 1H, NH), 8.23 (d, 1H, BIT C7-H), 8.05 (d, 1H, BIT C4-H), 7.75 (m, 2H, BIT C5,6-H), 7.25 (s, 1H, indole C7-H), 6.82 (s, 1H, indole C4-H), 3.95 (m, 4H, NCH), 3.45 (s, 2H, indole C3-H), 2.80 (m, 2H, ArCH), 2.70–2.50 (m, 6H, NCH)]. ZIP-SO2. A mixture of BITP-SO2 (81 mg, 0.32 mmol), OX-Cl (73 mg, 0.32 mmol), Na2CO3 (100 mg, 0.96 mmol), and KI (5 mg, 0.03 mmol) in 1.0 ml of acetonitrile was stirred and heated to reflux for 32 hr. Work-up of the reaction mixture as described for ZIP-SO gave the desired product as a pale yellow solid (22 mg). It was converted to the hydrochloride salt by stirring with 3 N HCl, filtering the solids, triturating with acetonitrile, and drying under vacuum at 70°C overnight to a pale pink solid [m.p. 295–297°C; FAB mass spectrum: m/z 447 (Cl), 445 (MH); H-NMR (dimethylsulfoxide-d6): d 10.45 (s, 1H, NH), 8.22 (dd, 1H, BIT C7-H), 8.04 (dd, 1H, BIT C4-H), 7.74 (m, 2H, BIT C5,6-H), 7.23 (s, 1H, indole C7-H), 6.78 (s, 1H, indole C4-H), 3.95 (m, 4H, NCH), 3.45 (s, 2H, indole C3-H), 2.84 (m, 2H), 2.70–2.50 (m, 6H, NCH)]. Animals. LE rats were purchased from Charles River Laboratories (Stoneridge, NY). Animals were quarantined for a minimum of 7 days before treatment and were maintained on a 12-hr light/dark cycle. Animals were fed food and water ad libitium and maintained with United States Department of Agriculture guidelines for the care and use of laboratory animals. Urinary, Biliary, and Fecal Excretion Studies. A group of three male and three female LE rats (240–260 g) were implanted with a PE-10 cannula into the common bile duct under anesthesia (sodium phenobarbital via ip injection). The animals were housed individually in stainless steel metabolic cages and were allowed to recover overnight before drug administration. The animals were orally administered 10 mg/kg radiolabeled ZIP. The dose was composed of a mixture of Hand C-labeled ZIP diluted with unlabeled drug to specific activities of 3.72 mCi/mmol and 2.82 mCi/mmol, respectively, and was administered as a suspension in 0.5% methyl-cellulose at a concentration of 1.5 mg/ml. Each animal received ;23 mCi of H-labeled and ;17 mCi of C-labeled material. All animals were fed at 2.5 hr after the dose and received electrolyte Krebs-Ringer solution throughout the study. Urine, bile, and feces from each animal were quantitatively collected for 7 days (168 hr after the dose) at 0–8, 8–24, 24–48, 48–72, 72–96, 96–120, 120–144, and 144–168 hr after the dose; the first feces sample was collected at 0–24 hr after the dose. The volumes of urine and bile samples were recorded, and all of the biological samples were stored at 220°C until analysis. Plasma Time Course Study. Another group of LE rats (N 5 14/gender, 270–320 g) were dosed by gavage with a 10 mg/kg dose of a mixture of Hand C-labeled ZIP. Blood was collected in heparinized tubes, by decapitation of two male and two female rats, at 0, 0.5, 1, 2, 4, 6, 8, 12, and 24 hr after the dose. The blood samples were centrifuged at 1000 g for 10 min to obtain the plasma. Samples were transferred to clean tubes and stored at 220°C until analysis. Determination of Radioactivity. Total radioactivity in urine, bile, and plasma was quantitated by counting sample aliquots (20–50 ml) using a H/C “dual-label” program with a Packard 2500 TR liquid scintillation counter. Ecolite-(1) scintillation cocktail (5 ml; ICN) was used for determination of the radioactivity in the samples. Quench curves were prepared separately for [H]ZIP and [C]ZIP. Fecal samples were lyophilized overnight and homogenized on a paint can shaker (Red Devil, model 5410), using chrome-plated 3/8 and 7/16 ball bearings. A small aliquot (20–60 mg) was combusted using a Packard Tricarb oxidizer (Irvine, CA). The liberated CO2 and H2O were trapped, and the radioactivity in the trapped samples was determined by counting in the liquid scintillation counter. Combustion efficiencies were determined by combusting C and H standards in an identical manner. The samples obtained at 0 hr after the dose were used as controls and counted to obtain a background count rate. Pharmacokinetic Analysis. Plasma concentrations of the unchanged ZIP were determined by a validated HPLC/MS/MS assay (22). Pharmacokinetic parameters were determined by standard methods. The AUCs were calculated from plasma concentrations of ZIP and total radioactivity, using a trapezoidal approximation of area and using 0 as the time 0 concentration. The tmax value was the time of the first occurrence of the maximal plasma concentration. Extraction of Metabolites from Biological Samples. Urine (1 ml) from each animal was centrifuged, and small aliquots (50 ml) were injected into the HPLC system without further purification. Bile (1 ml) was diluted with 4 ml of acetonitrile, and the precipitated protein was removed by centrifugation. The pellet was washed with an additional 2 ml of acetonitrile, and the two supernatants were combined. Small aliquots of supernatant and pellet were counted. The supernatant was concentrated and dissolved in 1 ml of mobile phase, and an aliquot (50 ml) was injected into the HPLC system. HPLC. HPLC was carried out on a system that consisted of a Rheodyne injector for manual injections, a LDC/Milton Roy constametric CM4000 gradient pump, a Waters Lambda-Max model 481 UV detector, a b-RAM, and a SP 4200 computing integrator. Chromatography was carried out on a YMC basic HPLC column (4.6 mm 3 250 mm, 5 mm) with a binary mixture of 20 mM ammonium acetate (pH 5.0, solvent A) and methanol (solvent B). The mobile phase initially consisted of solvent A/solvent B at 90:10 for 10 min. It was then linearly programmed to solvent A/solvent B at 20:80 over 50 min. Chromatography was carried out under isocratic conditions for 7 min and then programmed back to the starting solvent mixture over 8 min. The system was allowed to equilibrate for approximately 10 min before the next injection was made. The retention times of radioactive peaks were compared with those of the synthetic standards. FIG. 1. Structures of Cand H-labeled ZIP. 207 BIOTRANSFORMATION OF A BENZISOTHIAZOLYLPIPERAZINE at A PE T Jornals on Jne 2, 2017 dm d.aspurnals.org D ow nladed from Quantitative Assessment of Metabolite Excretion. Quantification of the metabolites was carried out by measuring radioactivity in the individual peaks that were separated on HPLC, using a b-RAM. The b-RAM provided an integrated printout in dpm and percentage of the radiolabeled material, as well as peak representation. The b-RAM was operated in the homogeneous liquid scintillation counting mode, with addition of 4 ml/min Ecolite scintillation cocktail to the eluent after UV detection. For simultaneous monitoring of Hand C-labeled compounds, efficiencies of 37% for H and 55% for C were used, with a compensation for C spill-over into the H window of 31%. These parameters were determined through separate injections of singly labeled standards. Enzyme Hydrolysis. Pooled rat bile and urine samples (0–24 hr, 0.5 ml each) were adjusted to pH 5 with sodium acetate buffer (0.1 M) and treated with 2500 units of b-glucuronidase/sulfatase. The mixture was incubated in a shaking water bath at 37°C for 12 hr and diluted with acetonitrile. The precipitated protein was removed by centrifugation. The pellet was washed with an additional 2 ml of acetonitrile, and the two supernatants were combined. The supernatant was concentrated and dissolved in 0.5 ml of mobile phase, and an aliquot (50 ml) was injected into the HPLC system. Incubation of bile and urine samples without the enzyme served as a control. Derivatization. One polar metabolite, M4, was isolated by HPLC and methylated with diazomethane. The purified metabolite M4 (100–200 ng) was dissolved in methanol (100 ml), and freshly prepared ethereal diazomethane (200 ml) was added. After standing for 15 min at room temperature, the solvent was removed with a stream of nitrogen and the residue was dissolved in the HPLC mobile phase. MS. Analysis of the metabolites was performed on a Perkin-Elmer (Norwalk, CT) SCIEX API III HPLC/MS/MS system using ion spray. The effluent from the HPLC column was split and about 50 ml/min was introduced into the atmospheric ionization source via an ion spray interface. The remaining effluent was directed into the flow cell of the b-RAM. The b-RAM response was recorded in real time by the mass spectrometer data system, which provided simultaneous detection of radioactivity and MS data. The delay in response between the two detectors was about 0.2 min, with the mass spectrometric response being recorded first. The ion spray interface was operated at 6000 V, and the mass spectrometer was operated in the positive mode. CID studies were performed using argon gas at a collision energy of 25–28 eV and a collision gas thickness of 3.5 3 10 molecules/cm.