Metabolism, Pharmacokinetics, and Excretion of the 5-Hydroxytryptamine 1B Receptor Antagonist Elzasonan in Humans S

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1 upplementl mteril to this rticle cn e found t: /1/ $2. DRUG METABLIM AD DIPITI Vol. 38, o. 11 Copyright 21 y The Americn ociety for Phrmcology nd Experimentl Therpeutics 34595/ DMD 38: , 21 Printed in U..A. Metolism, Phrmcokinetics, nd Excretion of the 5-ydroxytryptmine 1B Receptor Antgonist Elzsonn in umns Amin Kmel, 1 R. cott ch, Kevin Colizz, 1 Weiwei Wng, Thoms. Connell, Richrd V. Coelho, Jr., Ryn M. Kelley, nd Kls childknegt Deprtments of Phrmcokinetics, Phrmcodynmics nd Metolism (A.K., K.C., R..., W.W.), Explortory Medicinl ciences (T...), nd Reserch-API, Phrmceuticl ciences (R.V.C., R.M.K., K..), Pfizer Glol Reserch nd Development, Groton Lortories, Pfizer Inc., Groton, Connecticut Received My 19, 21; ccepted July 28, 21 ABTRACT: Introduction elective serotonin reuptke inhiitors re thought to provide symptomtic relief from depression y locking the reuptke of serotonin [5-hydroxytryptmine (5-T)] in the synptic cleft, therey correcting deficits in 5-T neurotrnsmission (lvey nd krepnek, 28; Cshmn et l., 29). imilr to selective serotonin reuptke inhiitors, 5-T 1B ntgonist is lso expected to elevte 5-T tone ut would do so y locking the terminl utoreceptor tht negtively Prts of this work were previously presented t the following conference: Kmel A, Wng W, Colizz K, nd Connell T (28) Metolism nd excretion of the 5-T 1B ntgonist [ 14 C]CP-448,187 in humns: determintion of the site of hydroxyltion of the mjor fecl metolite nd structurl chrcteriztion of the novel cyclized indole mjor circulting metolite y 1 LC-MR nd chemicl pproches. 56th AM Conference on Mss pectrometry nd Allied Topics; 28 Jun 1 8; Denver, Colordo. Americn ociety for Mss pectrometry, nt e, M. 1 Current ffilition: Metolism nd Phrmcokinetics, ovrtis Institutes for Biomedicl Reserch, Cmridge, Msschusetts. Article, puliction dte, nd cittion informtion cn e found t doi:1.1124/dmd The online version of this rticle (ville t contins supplementl mteril. The metolism, phrmcokinetics, nd excretion of potent nd selective 5-hydroxytryptmine 1B receptor ntgonist elzsonn hve een studied in six helthy mle humn sujects fter orl dministrtion of single 1-mg dose of [ 14 C]elzsonn. Totl recovery of the dministered dose ws 79% with pproximtely 58 nd 21% of the dministered rdioctive dose excreted in feces nd urine, respectively. The verge t 1/2 for elzsonn ws 31.5 h. Elzsonn ws extensively metolized, nd excret nd plsm were nlyzed using mss spectrometry nd MR spectroscopy to elucidte the structures of metolites. The mjor component of drug-relted mteril in the excret ws in the feces nd ws identified s 5-hydroxyelzsonn (M3), which ccounted for pproximtely 34% of the dministered dose. The mjor humn circulting metolite ws identified s the novel cyclized indole metolite (M6) nd ccounted for 65% of the totl rdioctivity. A mechnism for the formtion of M6 is proposed. urthermore, metolism-dependent covlent inding of drug-relted mteril ws oserved upon incution of [ 14 C]elzsonn with liver microsomes, nd dt suggest tht n indole iminium ion is involved. verll, the mjor metolic pthwys of elzsonn were due to romtic hydroxyltion(s) of the enzylidene moiety, -oxidtion t the piperzine ring, -demethyltion, indirect glucuronidtion, nd oxidtion, ring closure, nd susequent rerrngement to form M6. modultes 5-T relese (lssi, 22). Elzsonn (4-(3,4-dichloro- phenyl)-2-[2-(4-methyl-piperzin-1-yl)-enzylidene]-thiomorpholin-3- one; CP-448,187) exhiits potent nd selective ntgonism of 5-T 1B receptors in vitro, nd preclinicl in vivo studies demonstrte enhnced 5-T neurotrnsmission. The metolism nd phrmcokinetic of elzsonn were studied in niml species (Colizz et l., 28; Kmel et l., 29), nd results indicted tht most of the metolic pthwys were qulittively similr to those of humns. owever, the formtion of the novel cyclized indole metolite ws detected t considerly lower levels in the plsm of preclinicl species thn in humns. There re no known circulting phrmcologiclly ctive metolites of elzsonn, including the cyclized indole metolite. The present study ws undertken to ssess the metolic profile, the routes of excretion, nd the phrmcokinetics of elzsonn in helthy mle volunteers fter single dose of 1 mg of [ 14 C]elzsonn. The metolites of elzsonn were tenttively identified y ionspry LC-M/M using collision-induced dissocition (CID), neutrl loss, totl ion current, nd precursor ion scnning techniques. MR spectroscopy nd chemicl pproches were lso used to identify the mjor humn fecl nd circulting metolites. urthermore, n investigtion of unique cyclized indole metolite nd its down- Downloded from dmd.spetjournls.org t APET Journls on eptemer 27, 218 ABBREVIATI: 5-T, 5-hydroxytryptmine, serotonin; CP-448,187, 4-(3,4-dichloro-phenyl)-2-[2-(4-methyl-piperzin-1-yl)-enzylidene]-thiomorpholin-3-one, elzsonn; LC, liquid chromtogrphy; M/M, tndem mss spectrometry; CID, collision-induced dissocition; PLC, high-pressure liquid chromtogrphy; r, recominnt; CE-244,6, [4-(3,4-dichloro-phenyl)-2-[5-hydroxy-2-(4-methyl-piperzin-1-yl)-enzylidene]-thiomorpholin-3-one]; CP-69,77, elzsonn -oxide; CP-459,326, -desmethylelzsonn; AUC, re under the plsm concentrtiontime curve; ARC, ccurte rdioctivity counting; P45, cytochrome P

2 METABLIM AD DIPITI ELZAA C * strem iminium ion metolite nd role in in vitro covlent inding to liver microsoml protein is reported. Mterils nd Methods Generl Chemicls. Commercilly otined chemicls nd solvents were of PLC or nlyticl grde. Lun PLC columns were otined from Phenomenex (Torrnce, CA). Ecolite( ) scintilltion cocktil ws otined from MP Biochemicls (olon, ). Crosor nd Permfluor scintilltion cocktils were purchsed from Pckrd Instrument Compny (Downers Grove, IL). -Glucuronidse from elix pomti (type -1 with sulftse ctivity) ws otined from igm-aldrich (t. Louis, M). Recominnt (r) CYP3A4 nd CYP1A2 were purchsed from BD Gentest (Wourn, MA). Rdioleled Drug nd Reference Compounds. [ 14 C]Elzsonn (free se, ig. 1), leled t the 2-position of the 2-(4-methyl-piperzin-1-yl)- enzylidene moiety ttched to the thiomorpholin-3-one ring ws synthesized y the rdiochemistry group t Pfizer Glol Reserch nd Development (Groton, CT). [ 14 C]Elzsonn hd specific ctivity of mci/mmol nd rdiochemicl purity of 99% s determined y PLC with on-line rdioctivity detection. 5-ydroxyelzsonn ([4-(3,4-dichloro-phenyl)-2- [5-hydroxy-2-(4-methyl-piperzin-1-yl)-enzylidene]-thiomorpholin-3-one]; CE-244,6), M6 [4-(3,4-dichlorophenyl)-2-(2-methyl-1,2,3,4-tetrhydropyrzino[1,2-] indol-1-yl)thiomorpholin-3-one], elzsonn -oxide (CP-69,77), nd -desmethylelzsonn (CP-459,326) were synthesized y the Medicinl Chemistry group t Pfizer Glol Reserch nd Development nd served s synthetic stndrds to confirm the structures. A detiled description of the synthesis of rdioleled elzsonn nd metolite M6 re included in the supplementl dt. Urine, eces, nd Plsm Collection Methods. A mss lnce nd excretion study ws conducted in six helthy mle humn sujects. umn sujects were dministered single 1-mg (free se) orl dose of elzsonn contining 1 Ci of [ 14 C]elzsonn. Urine nd feces were collected from the sujects in 24-h intervls for minimum of 7 dys to mximum of 29 dys postdose. The first urine smple ws collected t to 12 h postdose. All iologicl smples were stored t 2 C until nlysis. mple nlysis ws performed within dys fter the completion dte of the study, nd metolite profiles on pooled smples were otined. or plsm profiles nd metolite identifiction, lood smples were collected into heprinized Vcutiners t the following time points: 2, 8, 24, 48, 96, nd 192 h postdose. The lood smples were centrifuged t 4 C, nd plsm ws trnsferred to clen tues. Plsm smples were pooled for ech humn suject nd stored t 2 C until nlysis. Pooling Methods to tin Quntittive nd Qulittive Dt. It is essentil tht quntittive metolite profiling represents ccurte metolite percentges relevnt to the AUC (or C vg ) rther thn n ritrry plsm pool. Therefore, for quntittive profiling of metolites in circultion, pooling ws done ccording to the method wherey smples re pooled in proportion to the time intervl ech smple represents (milton et l., 1981; Rid et l., 1991; op et l., 1998). Plsm smples were pooled such tht t lest 8% of the totl rdiolel AUC ws represented. or susequent metolite identifiction, the most concentrted smples were used to void dilution of circulting drug-relted mterils. or quntittive profiling of metolites in excret (urine nd feces), smples were pooled from ech individul in proportion to the mount (weight or C l IG. 1. tructure of 14 C-leled CP-448,187 (elzsonn) with numering system., site of the 14 C rdiolel. volume) of excret in ech smpling period. or susequent metolite identifiction (qulittive nlysis), the most concentrted smples were used to void unnecessry dilution of metolites. Determintion of Totl Rdioctivity. Totl rdioctivity in urine nd plsm ws determined y counting smple liquots (5 1 l) using 14 C progrm on Wllc 149 liquid scintilltion counter (PerkinElmer Life nd Anlyticl ciences, Wlthm, MA). Ecolite( ) scintilltion cocktil (2 ml) ws used for the determintion of the rdioctivity in the smples. Quench curves were prepred using [ 14 C]elzsonn t different concentrtions. ecl smples t ech time point were suspended in 5 to 2 ml of wter sed on smple weight nd homogenized, nd the totl weights of the fecl homogente were recorded. Aliquots (9 115 mg, in triplicte) of the homogentes were ir-dried nd comusted in Pckrd TriCr xidizer (PerkinElmer Life nd Anlyticl ciences). The lierted 14 C 2 ws trpped in Crosor nd Permfluor scintilltion cocktils (15 ml; PerkinElmer Life nd Anlyticl ciences), nd the rdioctivity in the trpped smples ws determined y counting on liquid scintilltion counter. Comustion efficiencies were determined y comusting 14 C stndrds (pec-chec; PerkinElmer Life nd Anlyticl ciences) in n identicl mnner. The smples otined t predose were used s controls nd counted to otin ckground count rte. The rdioctivity in urine nd feces t ech smpling time ws expressed s the percentge of dose excreted in the respective mtrices t tht smpling time. Concentrtions of Totl Rdioctivity nd Elzsonn. Blood smples (1 ml) from ech suject (n 6) were collected into heprinized Vcutiners t the following time points:, 2, 3, 4, 6, 8, 12, 24, 48, 72, 96, 144, 192, 24, 288, 336, 384, 432, 48, 54, nd 552 h postdose nd were centrifuged. The resulting plsm smples were plced into clen tues for shipment nd susequent nlysis. Totl rdioctivity in plsm ws determined y counting 5- l liquots from ech smple in duplicte using 14 C progrm on Wllc 149 liquid scintilltion counter. Ecolite( ) scintilltion cocktil ( 18. ml) ws used for the determintion of rdioctivity in the smples. Totl rdioctivity in the plsm smples ws converted into nnogrm equivlents per milliliter sed on the specific ctivity (the mount of rdioctivity per unit mount of sustnce) received y ech ptient (1 Ci of 14 C/1 mg of elzsonn). Plsm concentrtions of the unchnged elzsonn (nnogrms per milliliter) were determined y vlidted PLC-M/M ssy (orthwest Bionlyticl, lt Lke City, Uth). In Vitro Covlent Binding Experiments. [ 14 C]Elzsonn (1 M; 58 mci/mmol) ws incuted with pooled humn, femle rt, nd dog liver microsomes (1. mg/ml protein) in totl volume of.8 ml of K 2 P 4 (1 mm, p 7.4) contining 3.3 mm Mg 2. Incutions were commenced with the ddition of ADP to finl concentrtion of 1.3 mm. Ech determintion ws mde in triplicte. In humn liver microsoml incutions, the effect of vrious model nucleophiles nd other fctors were exmined: KC (.1 mm), G (5 mm), lysine (1 mm), ketoconzole (1. M), or humn liver cytosolic frction (3 mg/ml). Incutions were conducted for 2 min t 37 C in shking wter th open to the ir. Rections were terminted y the ddition of 5 volumes of C 3 C (4 ml) to precipitte the microsoml protein. The terminted incution mixture ws vortex-mixed nd centrifuged for 5 min (Joun swinging ucket, 2 rpm). The superntnt ws removed, nd the protein pellet ws wshed four times with 4.8 ml of 2 -C 3 C (2:8) nd five times with 4 ml of C 3 C. Wshes consisted of vortex mixing of the pellet in tue contining metl wire to ssist in disruption of the pellet, followed y centrifugtion s ove. Wshing continued until there remined no more detectle rdioctivity in the superntnt. The finl pellet ws dissolved in 1 ml of 2 M, nd the resulting solution ws nlyzed y liquid scintilltion counting in 18 ml of True-Count. To test the cid liility of the covlently ound mteril, one determintion ws mde y initilly precipitting the pellet with.1 M 4. The iosynthesized [ 14 C]elzsonn indole iminium ion metolite (see elow) ws lso exmined for in vitro covlent inding to pooled humn liver microsomes. The [ 14 C]iminium ion (.18 M) ws incuted with pooled humn liver microsomes (1. mg/ml) in.8 ml of K 2 P 4 (1 mm, p 7.4) contining 3.3 mm Mg 2. Incutions were conducted in the presence or sence of ADP (1.3 mm), KC (1. mm), or G (1 mm). Incutions were conducted for 2 min t 37 C in shking wter th open to the ir. Rections were terminted y the ddition of 4 ml of C 3 C, nd protein pellets were wshed nd nlyzed s descried ove. Downloded from dmd.spetjournls.org t APET Journls on eptemer 27, 218

3 1986 KAMEL ET AL. Biosynthesis of the Elzsonn Indole Iminium Ion Metolite. The iminium ion metolite ws iosynthesized using recominnt heterologously expressed humn CYP3A4. Elzsonn (2 M) ws incuted with rcyp3a4 (1 mg/ml protein) nd ADP (1.3 mm) in 2 ml of K 2 P 4 (p 7.45, 1 mm) nd 3.3 mm Mg 2. The incution ws conducted t 37 C in shking wter th open to the ir for 3 min. The rection ws terminted y the ddition of 6 ml of C 3 C, nd the precipitted protein ws removed y centrifugtion. The superntnt ws evported in TuroVp (Zymrk, opkinton, MA) t room temperture to volume of pproximtely.1 ml. The iminium ion ws purified y PLC. The smple ws injected onto Lun C18 column ( mm, 5 m; Phenomenex) equilirted in 1 mm 4 Ac contining 5% C 3 t flow rte of.8 ml/min. This condition ws held for 5 min followed y liner grdient to 8% C 3 over 2 min nd holding t this solvent composition for 5 min. rctions were collected ech minute. The effluent ws monitored with UV detection t 353 nm. The frction eluting t 23.5 min ws nlyzed y direct injection into ciex API1 mss spectrometer nd identified s the iminium ion metolite. The frction ws stored in the freezer in n mer vil. Reinjection of this frction on PLC-M system the following dy demonstrted tht the iminium ion ws stle. A smple of [ 14 C]iminium ion ws iosynthesized nd purified in the sme mnner, using [ 14 C]elzsonn s the sustrte. The moleculr ion of the [ 14 C]iminium ion ws m/z 446, consistent with the enrichment of one 14 C tom. Phrmcokinetic Anlysis. Phrmcokinetic prmeters were determined using the Winonlin noncomprtmentl nlysis computer progrm (version 3.2; Phrsight, Mountin View, CA). The res under the elzsonn nd totl rdioctivity AUCs were clculted from plsm concentrtions of elzsonn nd totl rdioctivity using liner trpezoidl pproximtion of re with zero s the time concentrtion. The mximum oserved plsm concentrtion (C mx ) ws estimted directly from experimentl dt, nd time to mximum plsm concentrtion (T mx ) ws defined s the time of first occurrence of C mx. The terminl elimintion phse rte constnt (K el ) ws estimted using lest-squres regression nlysis of the plsm elzsonn concentrtion-time dt otined during the terminl log-liner phse. The terminl phse hlf-life (t 1/2 ) ws clculted s ln(2)/k el, wheres the men t 1/2 ws estimted s ln(2)/men K el. Extrction of Metolites from Biologicl mples. Urine. Urine smples were pooled y suject s descried ove. The pooled smples were vortexmixed, nd 5 ml from ech pool ws evported to dryness in TuroVp. The residue ws reconstituted in 2 l of 1 mm mmonium cette (p 4.)- methnol-dimethyl sulfoxide (1.5:1.5:1). Aliquots (1 l) were directly injected without further purifiction onto the LC-ccurte rdioisotope counting (ARC) system (descried elow) for metolite profiling. or metolite identifiction, pproximtely 8-ml urine pools were dried in the TuroVp t room temperture nd then reconstituted in 6 l of 1 mm mmonium cette (p 4.)-methnol-dimethyl sulfoxide (1.5:1.5:1). Aliquots (9 l) were injected onto the PLC-M/M system. The LC system is descried elow. eces. ecl smples were pooled ccording to smple weight s descried ove. or metolite profiling, pproximtely 1 g of ech smple pool ws extrcted with 7 ml of cetonitrile- 2 (6:1 v/v) twice; 1- l liquots of oth extrctions were counted to determine recovery. Approximtely 87% of the rdioctivity ws recovered in the superntnts. The superntnt ws evported in TuroVp t room temperture, nd the residue ws reconstituted in 5 l of 1 mm mmonium cette (p 5.)-methnol-dimethyl sulfoxide (1.5:1.5:1). Aliquots (1 l) were injected onto the LC-ARC system for profiling. or metolite identifiction, 1 g of ech smple pool ws extrcted with 3 ml of cetonitrile- 2 (5:1 v/v) twice. The superntnts were comined nd dried in TuroVp t room temperture. The dried residue ws dissolved in 5 ml of wter nd extrcted with 15 ml of hexne twice. The hexne lyer ws removed, nd the queous lyer ws further extrcted with 15 ml of ethyl cette twice. The extrctions were comined nd dried in the TuroVp t room temperture, nd the residue ws reconstituted in 36 l of 1 mm mmonium cette (p 5.)-methnol-dimethyl sulfoxide (1.5:1.5:1). Aliquots of 9 l were injected onto the PLC-M/M system for metolite identifiction. Plsm. Plsm smples were pooled y humn suject seprtely ccording to the method descried ove. Aliquots (1 ml) from ech pool were extrcted with 5 ml of cetonitrile. The mixtures were vortex-mixed for 3 min, sonicted for 2 min, nd then centrifuged for 5 min t 2 C. Aliquots of the superntnt (two 5- l liquots) were counted to determine recovery. Approximtely 95% of the rdioctivity ws recovered in the superntnts. The superntnts were comined ccording to suject numer to two equl sets nd dried in the TuroVp t room temperture. ne set of the dried residue (six sujects) ws reconstituted in 15 l of 1 mm mmonium cette (p 4.)-methnol-dimethyl sulfoxide (1.5:1.5:1), nd liquots of 1 l were injected onto LC-ARC system for profiling. The other sets of dried residues were comined nd reconstituted in 3 l of 1 mm mmonium cette (p 4.)/methnol-dimethyl sulfoxide (1.5:1.5:1), nd 9 l ws injected onto PLC-M/M system for metolite identifiction. Quntittive Assessment of Metolites Using Rdiometric Detecting Method. Quntifiction of the metolites ws performed y mesuring rdioctivity in the individul PLC-seprted peks using -RAM instrument (I/U ystems, Tmp, L). The -RAM provided n integrted disply in counts per minute nd the percentge of the rdioleled mteril s well s pek representtion. The -RAM ws operted in the homogeneous liquid scintilltion counting mode with ddition of 4 ml/min of Ecolite( ) scintilltion cocktil to the eluent fter UV detection. Urinry, fecl, nd plsm profiles were quntittively very similr over time for t lest 12 months, indicting smple stility in these mtrices. Enzyme ydrolysis. Pooled humn urine smples ( 1 ml) were djusted to p 5 with sodium cette uffer (.1 M) nd treted with 25 units of -glucuronidse/sulftse. The mixture ws incuted in shking wter th t 37 C for 12 h nd ws diluted with cetonitrile ( 1 ml). The precipitted protein ws removed y centrifugtion. The pellet ws wshed with n dditionl 2 ml of cetonitrile, nd oth superntnts were comined. The superntnt ws concentrted nd dissolved in.5 ml of moile phse, nd n liquot (5 l) ws injected on the PLC column. Incution of the urine smples without the enzyme served s control. Reduction of -xide Metolite with Ti 3. The extrcted humn plsm smples or the dried humn urine smples were reconstituted in 1 l of 1 mm mmonium cette (p 4.)-methnol-dimethyl sulfoxide (1.5: 1.5:1). Titnium chloride solution (titnium chloride-2% hydrochloric cid; Thermo isher cientific, Wlthm, MA) ws dded drop y drop until the pink color of the rection mixture persists. Then 1 l of the rection mixture ws injected onto the LC-M system. mple Preprtion for 1 MR Anlysis of the Mjor umn ecl nd Plsm Metolites: Mjor umn ecl ydroxylted Metolite M3. Ten-grm liquots of pooled humn feces were extrcted twice with 7 ml of wter-cetonitrile (1:6, v/v). This process ws repeted for totl of eight extrctions to ensure dequte mteril for spectroscopic chrcteriztion. The superntnts were dried in TuroVp. The dried smples were redissolved in 5 ml of wter, extrcted with 5 ml of hexne twice (hexne extrcts were discrded), nd then extrcted with 5 ml of ethyl cette twice. The ethyl cette extrcts were comined nd dried in TuroVp for purifiction. PLC Purifiction of M3. orml-phse PLC ws used for the purifiction of M3. Chromtogrphy ws performed on Zorx il PLC column ( mm, 5 m) with inry mixture of hexne (solvent A) nd ethnol (solvent B). A flow rte of 1 ml/min ws used for the isoltion nd purifiction of M3. The moile phse initilly consisted of solvent A-solvent B (75:25, v/v) for 15 min. It ws then linerly progrmmed to 1% over 1-min period. The system ws operted isocrticlly for 4 min nd then linerly progrmmed ck to the initil condition in 1 min nd equilirted for 4 min. The dried ethyl cette extrcts residue ws loded into the PLC/ -RAM system to determine the retention time of M3 under the PLC condition descried ove. Injections were then loded into the PLC/UV system (LDC Anlyticl; Thermo Instrument ystems Inc., nt e, M), nd M3 frctions were collected, comined, nd dried in TuroVp nd finlly frozen t 2 C for 1 MR nlysis. Lrge-cle Biosynthesis of Mjor umn Circulting Metolite (M6). In vitro experiments using vrious sucellulr frctions s well s recominnt P45 to generte M6 in enough quntity for MR nlysis were unsuccessful. Therefore, unique pproch ws tken to generte M6 vi chemicl reduction of the iosynthesized iminium ion metolite. The rection mixture consisted of 9 l of stock [ 14 C]elzsonn (2 M) (1 g/ l in 5:5 Me- 2 ), 36 l of rcyp3a4 (9.6 mg of protein/ml, 19 pmol of Downloded from dmd.spetjournls.org t APET Journls on eptemer 27, 218

4 METABLIM AD DIPITI ELZAA 1987 P45/mg of protein, finl concentrtion 37 nm), 755 l of rcyp1a2 (8.6 mg of protein/ml, 38.4 of pmol of P45/mg of protein, finl concentrtion 25 nm), nd ml of phosphte uffer (p 7.4). The rection ws initited with 1 ml of cofctor solution (4 mg of -ADP, 16 mg D,L-isocitric cid ml of 125 mm Mg l of isocitric cid dehydrogense). This gve finl incution volume of 1 ml. The incution ws performed in 37 C for 1 h. or MR nlysis, the sme set of experiments ws performed using unleled elzsonn. After incution, the rection mixture ws divided eqully into 5 tues (2. ml in ech tue) nd stopped y the ddition of 5. ml of methyl tert-utyl ether to ech tue. Ech tue ws vortex-mixed using Multivortex mixer, centrifuged for 6 min, nd then flsh frozen on methnol with dry ice. The superntnts were comined nd dried in TuroVp. Quntittive ssessment of the reduction of elzsonn--oxide ck to elzsonn suggested tht CYP1A2 my e responsile for this reductive pthwy (Kmel et l., 29). Therefore, CYP1A2 ws dded to the incution mixture to lock or minimize the oxidtive pthwy for the formtion of the mjor -oxide metolite nd thus drive the rection to increse the yield for the formtion of the indole iminium ion metolite. This process proved to e successful to otin much lrger quntity of elzsonn indole iminium ion metolite. B 4 Reduction. Elzsonn indole iminium ion metolite ws isolted nd purified from the lrge-scle incuted smple using PLC. The dried indole iminium ion smple ws reconstituted in 2 l of nhydrous methnol nd pproximtely 2 g of B 4 (98%; Aldrich Chemicl Compny, Milwukee, MI) ws dded. The rection ws performed on ice for 3 min nd dried in the TuroVp. The sme experiment ws performed for unleled elzsonn. PLC Purifiction of the Reduced Indole Iminium Ion Metolite. The B 4 reduced smple ws reconstituted in 8 l of1mm 4 Ac (p 5)-Me- 2 t the rtio of 1.5:1.5:1.. Multiple 1- l liquots were then injected onto n PLC system tht consisted of P-15 utosmpler nd P-15 grdient pump. Chromtogrphy ws performed on Lun PLC column [ mm, 5 m, C18(2); Phenomenex] using inry mixture of 1 mm mmonium cette, p 5, with cetic cid (solvent A) nd 5:5 cetonitrile-c 3 (solvent B). A flow rte of 1. ml/min ws used. The moile phse initilly consisted of solvent A-solvent B (6:4) for 4-min period. It ws then linerly progrmmed to chieve mixture of solvent A-solvent B (3:7) over 15-min period. The system ws operted isocrticlly for nother 3 min nd then linerly progrmmed to chieve mixture of solvent A-solvent B (1:9) over 3-min period. The system ws operted isocticlly for nother 3 min nd then returned to the initil mixture of solvent A-solvent B (6:4) over 2-min period nd remined for nother 1 min. This gve totl run time of 4 min. The frction t retention time 24.2 to 25.2 min where M6 ws eluted ws collected, comined, dried in TuroVp, nd stored t 2 C for LC-MR nlysis. p Titrtion Curve nd Rection of the Iminium Ion Metolite with Cynide nd G. The indole metolite (2 M) ws incuted with mle monkey liver microsomes (2 mg/ml), ADP (1.3 mm), nd Mg 2 (3.3 mm) in 2 ml of K 2 P 4 t 37 C for 4 min to iosynthesize the iminium ion metolite. The incution ws terminted with C 3 C (2 ml) nd spun in centrifuge t 17g. To the superntnt ws dded 1 ml of.1% C, nd the solution ws spun in centrifuge t 4,g. The entire superntnt ws pplied to Polris C18 column ( mm; 5 m) t flow rte of.8 ml/min. The iminium ion metolite ws eluted using grdient eginning with.1% C in 3% C 3 C, which ws held for 5 min followed y liner increse in C 3 C to 6% t 5 min. The frctions eluting etween 2. nd 21.5 min contined the iminium ion nd were pooled nd used in susequent experiments. Becuse of its conjugted nture, the iminium ion metolite possesses strong sornce t 36 nm. To determine the p t which the iminium is converted to crinolmine, portion of the isolted frction ws exmined for sornce etween 5 nd 2 nm while the p ws incresed with K 3 P 4. Rections etween the iminium ion nd cynide ion nd with reduced G were exmined in solutions contining.5 M K 2 P 4 t p 7.5 y monitoring the sornce spectrum. PLC-ARC for the eprtion of All Plsm Metolites. PLC-ARC provides severl dvntges over trditionl detection systems (ssr et l., 23; Wng et l., 29) nd ws used for plsm profiling. The PLC-ARC TABLE 1 Rdioleled mss lnce of elzsonn in humns dministered 1-mg (free se) dose uject o. %Totl 14 C Excreted from 29 Dys Postdose Urine eces Totl M M M M M M Men D system consisted of n P 11 inry pump with n Agilent 11 utosmpler, n ARC stop-flow system (AIM Reserch Co., ewrk, DE), nd -RAM detector. The flow cell (2.2 ml volume) nd cocktil were otined from AIM Reserch Co. The flow ws stopped in 1-s intervls nd counted for 6 s. The 1 s of flow (.17 ml) mixes with enough scintilltion cocktil to fill the cell for ny given stop-flow period. The ckground counting threshold ws 3 cpm. Chromtogrphy ws identicl to tht used for the purifiction of the reduced iminium ion metolite s descried ove. Mss pectrometry. Anlysis of metolites ws conducted with CIEX API III plus (Perkin-Elmer Life nd Anlyticl ciences, Thornhill,, Cnd) equipped with n ionspry source. The effluent from the PLC column ws split, nd pproximtely 5 l/min ws introduced into the tmospheric pressure ioniztion source. The remining effluent ws directed into the flow cell of the -RAM detector. The -RAM response ws recorded in rel time y the mss spectrometer dt system tht provided simultneous detection of rdioctivity nd mss spectrometry dt. The dely in response etween the two detectors ws pproximtely.2 min with the mss spectrometric response recorded erlier. The ionspry interfce ws operted t 5 V, nd the mss spectrometer ws operted in the positive mode. CID studies were performed using rgon gs t collision energy of 25 to 28 ev nd collision gs thickness of molecules/cm 2. MR. mples were nlyzed y n LC-M-MR system consisting of n Agilent 11 inry pump (Agilent Technologies, nt r, CA), Bruker Biopin BU- column oven (Bruker Biopin Corportion, Billeric, MA), Bruker Biopin photodiode rry detector, Bruker Biopin BMI interfce using 2:1 split, Bruker Dltonics Esquire 3 ion trp mss spectrometer (Bruker Dltonics, Billeric, MA) equipped with n electrospry source, Bruker Biopin BPU-36 pek storge unit, nd Bruker 5 Mz Avnce DRX spectrometer equipped with 4-mm 1-13 C inverse z-grdient LC flow proe. Anlyte within smple liquot (1 l) ws eluted t.5 ml/min over reverse-phse column (5 m, 3 15 mm) t room temperture using n queous uffer of (1 mm CD 3 CD in D 2 djusted to pd 4 with D 4 D) or (.1% C 3 CD in D 2 ) nd using CD 3 D or CD 3 C s orgnic eluent. ive percent of the column effluent ws split postcolumn nd diluted with 1% D 2 incd 3 C (contining.2% CD 3 CD) t flow rte of 125 l/min efore entering the mss spectrometer; the remining 95% of the effluent pssed through the diode rry detector. The peks of interest were stored in the BPU-36 unit using the loop storge technique nd susequently introduced into the MR spectrometer. 1 nd correltion spectroscopy spectr were otined on LC peks of interest using doule presturtion of solvent MR resonnces. Proton chemicl shifts re reported in prts per million reltive to tetrmethylsilne s referenced from the shift of residul protons in CD 3 C (1.94 ppm) or from the shift of residul protons in CD 3 D t 3.33 ppm. Results Excretion. The men percentge of totl rdioctivity recovered from to 29 dys in urine nd feces of six humn sujects fter orl dministrtion of [ 14 C]elzsonn is shown in Tle 1. The verge cumultive recovery in humn urine, feces, nd the sum of urine nd feces is depicted in ig. 2. Phrmcokinetics. The men plsm concentrtion-time curve for elzsonn (nnogrms per milliliter) nd totl circulting rdiocti- Downloded from dmd.spetjournls.org t APET Journls on eptemer 27, 218

5 1988 KAMEL ET AL. IG. 2. Men cumultive recovery of 14 C in humn urine nd feces nd totl recovery of rdioctivity ( 36 h postdose) fter orl dministrtion of 1 mg (free se)/kg of elzsonn (f, urine;, feces; Œ, totl). vity (nnogrm equivlents per milliliter) re grphiclly depicted in ig. 3. A comprison of the AUC t lst, t 1/2, nd C mx etween drug nd totl rdioctivity is shown in Tle 2. Metolic Profiles in Urine, eces, nd Plsm. Urine, fecl, nd plsm profiles otined for ll sujects were of similr qulity. Representtive PLC rdiochromtogrms of metolites in humn urine ( 96 h), feces ( h), nd plsm ( 96 h) fter orl dministrtion of [ 14 C]elzsonn re depicted in ig. 4. The urine profiles for ll sujects were qulittively similr. Qulittive similrities were lso oserved for ll fecl profiles. owever, there were some quntittive differences in the excretion of metolites in urine nd feces. Qulittive nd quntittive similrities of the circulting metolites were oserved for ll humn sujects. A summry of the undnce nd description of the mjor humn metolites in urine, feces, nd plsm is presented in Tle 3. In ddition to the prent drug, totl of two metolites in urine, four metolites in feces, nd three metolites in plsm were identified. M3, M5, nd M6 were the mjor metolites identified in feces, urine, nd plsm, respectively. Men Plsm Concentrtion (ng or ng(eq)/ml) Time (hour) Identifiction of Metolites. The structures of metolites were elucidted y ionspry LC-M/M, MR, nd LC-MR, s well s chemicl pproches or comprison to synthetic stndrds, when ville. Comined liquid chromtogrphy-ionspry mss spectrometry (full scn) nd tndem mss spectrometry, such s precursor ion, product ion, single ion monitoring, nd multiple rection monitoring scnning techniques were used for the identifiction of metolites. Where possile, the identities of metolites were confirmed y coelution on PLC with synthetic stndrds. The word tenttive ws used when the exct sites of some structurl modifiction could not e determined. Elzsonn hd retention time of 27.8 min on the PLC system nd showed protonted moleculr ion t m/z 448. The CID mss spectrum of elzsonn (ig. 5) showed frgment ions t m/z 433, 391, nd 348, which represent the dichloro-phenylenzylidene-thiomorpholinone moiety. The frgment ions t m/z 228, 199, nd 185 represent the piperzinyl-enzylidene moiety. The frgment ions t m/z 24 nd 144 represent the enzylidene moiety. Totl rdioctivity (ng. eq/ml) Drug (ng/ml) IG. 3. Men plsm concentrtions of elzsonn nd men totl rdioctivity for six humn sujects fter single orl dose of [ 14 C]elzsonn. Œ, totl rdioctivity (nnogrm equivlents per milliliter);, drug (nnogrms per milliliter). Error rs indicte.d. Downloded from dmd.spetjournls.org t APET Journls on eptemer 27, Time (hour)

6 METABLIM AD DIPITI ELZAA 1989 TABLE 2 Comprison of men vlues of AUC t lst,t 1/2, nd C mx for totl rdioctivity nd elzsonn in humn plsm fter orl dministrtion of 14 C elzsonn Dt re presented s mens.d. n 6. Men represents dt from ech suject. Eight nd 11 dt points were used to clculte t 1/2 for elzsonn nd totl rdioctivity, respectively. Vlue AUC t lst (ng h/ml) Elzsonn Totl rdioctivity Drug % of totl rdioctivity 12.3 t 1/2 (h) Elzsonn Totl rdioctivity C mx (ng/ml) Elzsonn C mx (ng-eq/ml) Totl rdioctivity Drug % of totl rdioctivity 63.2 The frgment ions t m/z 72 nd 7 were the most undnt ions in the elzsonn mss spectrum nd were derived from the methylpiperzine moiety. Prent drug nd totl of six metolites were identified. Unchnged drug ws found in urine, feces, nd plsm smples. The structures of these metolites were identicl to those identified previously in preclinicl species (Colizz et l., 28; Kmel et l., 29). M6 ws novel mjor circulting metolite nd required further structurl confirmtion. M1. M1 hd retention time of 21.6 min on the PLC-ARC system nd showed protonted moleculr ion t m/z 64, 192 mss units higher thn tht of the unchnged drug (m/z 448), suggesting tht it ws conjugte. M1 ws only detected in urine nd ccounted for 5.1% of the dose (verge of six humn sujects). Its CID spectrum (dt not shown) showed the frgment ion t m/z 464, loss of 176 mss units, suggesting tht M1 ws glucuronide conjugte. The frgment ion t m/z 464, 16 mss units higher thn the moleculr ion of unchnged drug, suggested the ddition of n oxygen tom to the molecule. The sence of wter loss (18 mu) in the CID spectrum of M1 suggested tht liphtic hydroxyltion hd not occurred. urther collision-induced dissocition of the frgment ion t m/z 464 (dt not shown) showed the frgment ion t m/z 215, indicting tht the oxidtion hd occurred remote from the dichloro-phenyl-thiomorpholinone moiety. The frgment ion t m/z 215, 16 D higher thn the frgment ion t m/z 199 of the unchnged drug, further suggested tht the enzylidene moiety ws the site of oxidtion. The exct position of the oxidtion could not e determined from mss spectrl dt. Tretment of humn urine metolites with -glucuronidse/sulftse resulted in the disppernce of M1 nd n increse in the response of unchnged drug in the rdiochromtogrm (dt not shown). Bsed on these dt, M1 ws tenttively identified s the glucuronide conjugte of the oxidtive product of elzsonn. M2. M2 hd retention time of 25.4 min on the PLC system nd showed protonted moleculr ion of m/z 464, 16 mss units higher thn tht of the prent drug, suggesting tht the molecule hd undergone monooxidtion. M2 ccounted for 6.2% of the dministered dose (verge of six humn sujects) in fecl smples nd ws not detected in urine or plsm. The CID mss spectrum (dt not shown) of M2 ws identicl to tht of M3 descried elow, nd the exct position of the oxidtion could not e determined from mss spectrl dt. Bsed on these dt, M2 ws identified s the hydroxylted product of the prent drug. M3. M3 hd retention time of 25.9 min on the PLC-ARC system nd lso showed protonted moleculr ion t m/z 464, 16 mss units higher thn tht of the prent drug, suggesting tht the molecule hd undergone monooxidtion. M3 ccounted for 34.4% of the dministered dose (verge of six humn sujects) in the fecl smples nd ws not detected in urine or plsm. Its CID spectrum is depicted in ig. 6 nd showed the frgment ion t m/z 449, loss of 15 mss units ( C 3 ), suggesting tht no modifiction hd occurred on the piperzine ring. The frgment ions t m/z 72, 7, nd 58 further suggested tht the piperzine ring ws intct. The frgment ions t m/z 244, 215, nd 19 indicted tht the oxidtion hd occurred remote from the dichloro-phenyl-thiomorpholinone moiety. The frgment ions t m/z 16 nd 146, 16 D higher thn the frgment ions t m/z 144 nd 13 of the unchnged drug, suggested tht the enzylidene moiety ws the site of oxidtion. The frgment ion t m/z 21, 16 D higher thn the frgment ion t m/z 185 of the unchnged drug, further suggested tht the enzylidene moiety ws the site of oxidtion. The exct position of the hydroxyltion ws determined from MR dt. igure 7 shows comprison of the romtic region of the 1 spectrum for the purified fecl hydroxylted metolite M3 (top) nd elzsonn (ottom). The 1 spectrum for M3 shows chnge in the romtic region, consistent with hydroxyltion t either position 5 or 6. The signls t 7.62 (d), 7.34 (dd), nd 7.67 (d) indicte tht the dichloro-phenyl moiety is unchnged. The singlet t 7.99 indictes tht the olefinic proton is unchnged. The remining romtic resonnces 7.12 (d), 6.89 (dd), nd 7.28 (d) re consistent with hydroxyltion t position 5. owever, sed on the metolite 1 spectrum lone, it is not possile to definitively rule out hydroxyltion t position 4. Comprison of the metolite 1 spectrum with spectrum of genuine synthetic stndrd CE-244,6 (dt not shown) further supported this structure ssignment nd indicted tht the two compounds hve the sme spectrl pttern nd therefore the sme chemicl structure. M3 ws coeluted with the synthetic stndrd on PLC nd hd n identicl CID mss spectrum (ig. 6). Bsed on these dt, M3 ws unequivoclly identified s 4-(3,4-dichloro-phenyl)-2-[5- hydroxy-2-(4-methyl-piperzin-1-yl)-enzylidene]-thiomorpholin-3- one. Unchnged drug (elzsonn). Elzsonn hd retention time of 27.8 min on the PLC-ARC system nd showed protonted moleculr ion t m/z 448. Prent drug ccounted for 5.5% of the dministered dose in fecl smples nd 5% in urine. Unchnged drug coeluted with synthetic stndrd on the PLC system nd hd n identicl CID spectrum. M4. M4 hd retention time of 27.9 min on the LC-ARC system nd showed protonted moleculr ion t m/z 434, 14 mss units lower thn the drug, suggesting tht the molecule hd undergone -demethyltion. M4 ccounted for 3.8% of the totl rdioctivity (verge of six humn sujects) in plsm nd ws not detected in urine or feces. M4 ws coeluted with the synthetic stndrd nd hd n identicl CID mss spectrum (dt not shown). Bsed on these dt, M4 ws identified s 4-(3,4-dichlorophenyl)-2-(2-piperzin-1- yl-enzylidene)-thiomorpholin-3-one. M5. M5 hd retention time of 3.5 min on the PLC-ARC system nd showed protonted moleculr ion of m/z 464, 16 mss units higher thn tht of the prent drug, suggesting tht the molecule hd undergone monooxidtion. M5 ccounted for 5.6% (M5 M6) of the dministered dose in fecl smples, 7.8% in urine, nd 5.5% of the totl rdioctivity in plsm (verge of six humn sujects). The CID mss spectrum of M5 (dt not shown) showed the frgment ion t m/z 447, loss of 17 D from m/z 464, suggesting tht the molecule hd undergone -oxidtion. The sence of wter loss (18 mss units) in the CID spectrum of M5 further suggested tht liphtic hydroxyltion hd not occurred. Tretment of humn urine nd plsm metolites with queous Ti 3 resulted in the dispper- Downloded from dmd.spetjournls.org t APET Journls on eptemer 27, 218

7 199 KAMEL ET AL. ivity (dpm) Rdiocti ivity (dpm) Rdiocti ctivity (dpm) Rdio Urine (suject #2) nce of M5 nd n increse of the response of unchnged drug in the rdiochromtogrm (dt not shown). The CID mss spectr of M5 from plsm nd urine were lso identicl to tht of the synthetic stndrd. Bsed on these dt, M5 ws identified s elzsonn - oxide. Indole iminium ion metolite. The indole iminium metolite ws not detected in humns nd showed moleculr ion t m/z 444, 4 D lower thn tht of the prent drug. Its CID spectrum (dt not shown) showed the mjor frgment ions t m/z 225 nd 229 nd suggested tht the prent drug hd undergone oxidtion followed y cycliztion. The frgment ion t m/z 225, 3 mss units lower thn the prent drug, suggested tht the nitrogen is chrged nd the oserved moleculr ion t m/z 444 is M, not [M ]. igure 8 shows comprison of the 1 spectrum of elzsonn (top) nd the indole iminium metolite (ottom). By inspection, it is cler tht there hs een mjor chnge to the metolite. There is shift in the -methyl resonnce from 2.82 M1 elzsonn Time (min) 9 eces (suject #6) M elzsonn M2 M5M Time (min) M5M Plsm (suject #5) M5 M4elzsonn Time (min) IG. 4. Representtive PLC rdiochromtogrms of urinry (suject 2, 96 h postdose pooled), fecl (suject 6, h postdose pooled), nd plsm (suject 5, 96 h postdose pooled) metolites of elzsonn fter single orl dministrtion of [ 14 C]elzsonn t dose of 1 mg. ppm in the elzsonn spectrum to 3.69 ppm in the indole iminium metolite spectrum, indictive of positive chrge nd consistent with quternry nitrogen. The correltion spectroscopy spectrum (dt not shown) shows correltion from this methyl resonnce t 3.69 ppm to new significntly downfield singlet t 9.2 ppm s well s to methylene resonnce t 4.15 ppm. In ddition, the singlet t 7.79 ppm in the elzsonn spectrum is missing in the indole iminium metolite spectrum, nd there is new singlet t 5.68 ppm. In ddition, there is significnt chnge in the overll UV profile (dt not shown) with shift in the UV mxim from 331 nm in the prent spectrum to 358 nm in the metolite spectrum, consistent with more conjugted chromophore in the metolite. These oservtions cn only e explined y the formtion of n indole iminium ion. M6. M6 coeluted with M5 nd hd retention time of 3.5 min on the LC-ARC system. M6 showed protonted moleculr ion t m/z Downloded from dmd.spetjournls.org t APET Journls on eptemer 27, 218 TABLE 3 Averge percentges of urinry, fecl, nd plsm metolites of elzsonn in humn sujects fter dministrtion of 1-mg (free dose) orl dose Dt re presented s mens.d. n 6. Metolite m/z Description Urine % Dose %Totl Rdioctivity in Plsm M1 64 ydroxyglucuronide D..D. M , 4-, or 6-.D D. M D D. Prent 448 Elzsonn M Desmethyl.D..D M xide (M5 M6) M6 446 Cyclized indole product.d (M5 M6) Totls D., not detected;, hydroxy. Clculted on the sis of totl % 14 C excreted from urine nd feces of ech suject shown in Tle 1 (dose metolite numer % rdioctivity metolite numer totl % 14 C excreted). Most likely position 3 on the sis of electronic considertion. eces

8 METABLIM AD DIPITI ELZAA 1991 sity (%) Reltive Intens C 2 C C 3 C 3 C 2 C C 2 C 446, 2 mss units lower thn the drug, nd 2 mss units higher thn the indole iminium ion metolite. Its CID spectrum (dt not shown) showed the se frgment ion exclusively t m/z 185. The CID mss spectrum of m/z 448 ( 37 ) of M6 lso showed the frgment ion t m/z 185 s the lmost exclusive product ion in the spectrum, suggesting tht this frgment ion contins no toms nd tht the molecule hd undergone ring closure nd rerrngement to form stle methyltetrhydro-pyrzino-indolylidene-like structure. Both the indole iminuim ion metolite nd M6 (very likely to e the precursor of the ity (%) Reltive Intensi ity (%) Reltive Intensi C 3 C C C C m/z 331 m/z m/z 448 [M ] 433 IG. 5. CID product ion mss spectrum of [M ] of elzsonn stndrd t m/z 448. indole iminium ion metolite) were iosynthesized on lrge scle, nd the iminium ion metolite ws isolted nd purified y PLC. Tretment of the purified indole iminium ion metolite with B 4 resulted in the disppernce of the indole iminium ion metolite nd the ppernce of new pek, which coeluted with M6 on PLC (ig. 9). A new PLC method ws developed with chromtogrphic seprtion for ll humn circulting metolites (ig. 1). The mjor humn plsm metolite M6 ws coeluted with the iosynthetic M6 on PLC nd hd n identicl CID spectrum. piking humn plsm 47 [M] m/z 449 [M] [M-Me] 464 IG. 6. CID product ion mss spectr of m/z 464 (M3) from humn fecl smple () nd synthetic stndrd (). Downloded from dmd.spetjournls.org t APET Journls on eptemer 27, 218

9 1992 KAMEL ET AL. 5--elzsonn e h f impurities g d e d f g h elzsonn ppm e d h f g 4 1 c e d f & c ppm IG LC-MR spectr of elzsonn nd hydroxylted metolite from humn feces. smple with the purified rdioleled iosynthetic M6 resulted in n increse of the rdioctive response of M6 from the humn plsm smple (dt not shown). Both M6 nd the chemiclly reduced indole iminium ion metolite (fter eing treted with B 4 ) hve identicl CID mss spectr (ig. 11). igure 12 shows comprison of the downfield region of the 1 spectr of elzsonn (top), the ove-identified indole iminium ion metolite (middle), nd metolite M6 (ottom) from in vitro humn incutions with rcyp3a4. It is cler y inspection tht there re significnt differences in the romtic moiety of these compounds. The singlet t 8 ppm in the elzsonn spectrum is missing in the two other spectr, indicting tht the olefinic proton t the enzylidene moiety is not present in either metolite. In ddition, the singlet t 5.7 ppm in the iminium ion spectrum is present in the metolite M6 spectrum t 5.6 ppm, indicting tht metolite M6 is similr cyclized compound yielding n indole moiety. In ddition, there is no pek t 9 ppm in the M6 spectrum s there is in the iminium ion spectrum, indicting tht the proton of the piperzine ring t position 3 is not djcent to n iminium ion. These oservtions re consistent with the proposed cyclized structure. A complete synthetic pthwy for M6 is descried in the supplementl dt nd ws used to confirm structure nd test M6 for iologicl ctivity. M6 ccounted (verge of six humn sujects) for 5.6% (sum of M5 nd M6) of the dministered dose in feces nd 65% of the totl rdioctivity in plsm nd ws not detected in urine. n the sis of the ove dt, M6 ws identified s 4-(3,4-dichlorophenyl)-2-(2- methyl-1,2,3,4-tetrhydropyrzino[1,2-]indol-1-yl)thiomorpholin- 3-one. In Vitro Covlent Binding of [ 14 C]Elzsonn nd Indole Iminium Ion Metolite. Upon incution of [ 14 C]elzsonn with humn liver microsomes, in the presence of ADP covlent incorportion into microsoml protein ws oserved (men pmol/mg of protein in 2 min). In the sence of ADP, little inding ws oserved. The ddition of model nucleophiles, lysine nd cynide, did little to reduce the inding; however, the ddition of reduced G resulted in 52% decrese in inding. The inding ws gretly reduced in the presence of ketoconzole, indicting tht CYP3A ctivity ws necessry to produce the intermedite tht covlently inds to microsoml protein. The ddition of cytosol, source of ldehyde oxidse (n enzyme tht converts licyclic iminium ions to lctms) did not cuse reduction in inding. Binding ws lso oserved in rt nd dog liver microsomes, leit to lesser extent. The inding of iminium ions to nucleophilic groups represents ond tht is reversile in cid. Microsomes tht were sujected to the sme incution conditions with [ 14 C]elzsonn, ut initilly precipitted with wek perchloric cid, demonstrted lower extent of covlent incorportion. The mount of covlent inding of rdioleled mteril to microsoml protein under vrious conditions is listed in Tle 4. Biosynthesized [ 14 C]elzsonn indole iminium ion ws exmined for metolism dependent nd independent of covlent incorportion into humn liver microsomes. In the sence of ADP, n verge of 1% of the rdioleled mteril ws incorported (1.9 pmol/mg of g h Downloded from dmd.spetjournls.org t APET Journls on eptemer 27, 218

10 METABLIM AD DIPITI ELZAA 1993 Elzsonn -Methyl D ppm Indole iminium ion metolite ) Rdio octivity (dpm Rd dioctivity (dp pm) D -Methyl ppm rcyp3a4 -desmethyl M4 Indole iminium ion -xide M5 elzsonn IG MR spectr of elzsonn (top) nd the indol iminium metolite (ottom). Indole cyclized Metolite Time (min) Indole minium ion metolite pek ws isolted, purified nd treted with B M6 p Titrtion Curve nd Rection of the Iminium Ion Metolite with Cynide nd G. The chemistry nd rectivity of the iminium ion metolite ws exmined. The equilirium etween iminium ions nd crinolmines will e dependent on the concen- IG. 9. PLC rdiochromtogrms of metolites of [ 14 C]elzsonn fter incution with humn rcyp3a4 (1 mm elzsonn, 5 pmol/ ml rcyp, 1-h incution). Downloded from dmd.spetjournls.org t APET Journls on eptemer 27, 218 Time (min) protein) (Tle 5). This inding ws prtilly reduced in the presence of KC or G. owever, n enhncement of incorportion of rdioctivity ws oserved in the presence of ADP, suggesting tht the iminium ion is further metolized to other rective intermedites.

11 1994 KAMEL ET AL. CPM (x1 2 ) elzsonn -desmethyl (M4)) -xide cyclized indol metolite M5M Bkg Rdioctivity (dpm) Reltive In tensity (%) Time (min) elzsonn -desmethyl M4 -xide M5 Indole cyclized Metolite M Time (min) m/z 185 [M] IG. 1. LC-ARC rdiochromtogrms of plsm (suject, 96 h postdose pooled) metolites of elzsonn efore () nd fter () chromtogrphic seprtion of ll metolites. IG. 11. CID mss spectrum of M6 t m/z 446 from humn plsm () nd the iminium ion metolite () fter eing isolted, purified, nd treted with B 4. Downloded from dmd.spetjournls.org t APET Journls on eptemer 27, 218 Rel tive Intensity (%) [M] m/z trtion of hydroxide ion nd hence on p. At cidic nd neutrl p, the iminium ion form is present, s oserved through the sornce mximum t 36 nm (ig. 13A). As the p ws incresed to 12.2, the sornce decresed, nd the inflection point of the titrtion ws t pproximtely p 1.5 (ig. 13B). The iminium ion ws shown to rect with cynide ion, s shown y the decrese in sornce t 36 nm in ig. 13C. owever, ttempts to isolte the -cynomine nd generte mss spectrl dt were unsuccessful. Mixing the iminium