g. keith mulholland et al- synthesis, rodent biodistribution, dosimetry, metabolism, and monkey...

8/3/2019 G. Keith Mulholland et al- Synthesis, Rodent Biodistribution, Dosimetry, Metabolism, and Monkey Images of Carbon

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binding in degenerative brain disorders such as Akhe imer 's , Hunting ton 's , and Park inson' s d isease(16).Forthesereasons ,herehasbeencon sider ablenterestn studyin g th e re gio na l d is trib ut io n o f c en tra l mu sc ari nic c ho li nergic binding sites in m an with labeled receptor bindingd ru gs a nd e xte rn al im ag in g te ch niq ue s. A n umbe r o f h ig haffin ity rad io lab eled m uscarin ic antago nists h av e b een p rep are d fo r in ve stig atio n a s p ote ntia l m usc arin ic im ag in gagents, including quinucidinyl benzilate (Q NB ) analogsla be le d w ith ra dio io din e ( 7 11and C12), dexetimidea na lo gs c on ta in in gra dio io din e(1 3), C(1 4),a nd 8 F,15 ,16), [ C] scopo lamine1 7 ,18) and [C]benztropine1921). There hasbeenparticularinterestin usingpositronem ission tom ography (PET) with an appropriate m uscarinic ligand because of the hope that this technique's highresolution and ability for noninvasive quantification ofradioactivity distribution in the living hum an brain willprovid e new inform ation concerning relation ships between mAChR activity and brain functional status ind isease an d in h ealth.In connection w ith ongoing efforts to study mAC hRw ith P ET (1 8,22), w e ha ve recently exam ined [ Ciscopolam ine in hum ans (23). The present w ork stem s fromour search for alternate mAC hR rad ioligands w ith m orefa vo ra ble tra ce r c ha ra cte ris tic s a nd mo re con venie nt s ynth eti c a cc es s t h an s co po lam in e (2 4). A p re lim in ary p art o fthis search involved in vitro and ex vivo screening of ag ro up o f cand id ate antim uscarm nic b en zilate ester lig and sin unlabeled form s for m uscarinic specificity, binding affinity and brain penetration (25). From this group ofcom pounds (+)2a-tropanyl benzilate (TRB) (Schem e 1),a potent centrally active mAC hR antagonist (26), w asselec ted for radiosynthesis and further study. H ere theb io lo gic al p ro pe rtie s o f [ C ]TRBa re exam in ed in r od en tsand nonhuman pr imates ,and some compar isons betweenthe rodent data of [ C]T RB and [1C jscopolam ine aremad e. T he se re su lts re pre se nt in te rm ed ia te s te ps towa rd

Muscanniccholmne rgiceceptors (mAC hA)are abunda ntinthe brain,andthe mAChR systemmediatesmanyaspectsofb ra in functi on . Ther e is evidence o f a lte ra tions in muscar in icbinding in degenerat ive brain disorders. A muscannic receptorradio l igand, carbon- i 1-(+)-2a-t ropanyl benzila te ( [11C]TRB),has been preparedthroughN-[11C]methylat ionf N-desmethyl TRB, and evaluatedin rodentsand pr imates .Fullbodybiodistnbutionn rats has been determinedand the expectedhumandosime trycalculated.Com parisonswith [11CJscopo laminein rats showed 26imesgreater brainuptakeof [11C]TRB . Highlyspecificand saturablebindingof [11C]TRBin thestnatumand cortexwas demonstratedby greater than 85%blockadeof uptakefollowingQNB or scopolaminepretreatment. S tri at um /cer ebe llum rat io s in m ice a t 60 mm exceeded12.6 .TLCanalys isfrattissu eshow edheabsencef11C -me tabo lite s in b ra in a nd hea rt, a nd a ra pid s olid p ha se C -i 8S ep -P ak m eth od fo un d th at u nm eta bo Iiz ed p la sm a [1 1C ]T RBin monkeysfe ll f rom 8 i % a t 5 mmto 48% a t 80 mm.F inally ,b ra in s o f liv in g p rim ate s h av e b ee n im ag ed u sin g PET a nd[11C]TRB;regional localization was consis tent wi th muscannicre ce pto r d istrib utio n. T he se re su lts re pre se nt in te rm ed ia tes te ps in th e dev elo pment o f [1 1C ]TRB f or q uantific atio n o fc en tra l m us ca nn ic re ce pto rs in m an .J Nuc IMed 1992 ;33 :423430

uscarmniccho linerg ic recep to rs (mAChR) are abund an t in th e b ra in , p art ic ula rly in s tria ta l a nd cor tic al a re as ,and the mAC hR system m ediates m any aspects of brainfunction, including higher processes of cognition andmemor y. The re is evidenceo f a lte ra tions in muscarmnic

Rec ei ve d May 1 5, 1 99 1; r ev is io n a cc ep te d O ct. 3 1, 1 99 1.For rep r in ts contac t: G . Kef thM uiho lland ,PhD, Ihi iVe rSf ty f Michigan ,D iv is io n o f Nudear Medi cin e, Cycl ot ro n/PET FaaI It y, 3 48 0 K re sg e I II , A nnArbor , MI48109 .

42 3ynthe sisand Eva luationof [11]TR Bulhollandet al

Synthesis , Rodent Biodistr ibution, Dosimetry,Metabolism , and Monkey Images of Carbon-11-Labeled (+)-2a-Tropany l Benzila te: A Centra lMuscarin ic Receptor Imaging AgentG. Keith Mu lholland ,* Char lo tte A . 0@0,t Doug la s M . J ewett,* M ichael R . K ilb ou rn ,* Rob er t A . Koeppe ,*P hillip S . S herm an,* N eil A . P etry,* Jam es E . C arey,* E dw ard R . A tk inson,@ S ydney A rcher,@K irk A . F rey* andD avid E . K uhl*4D jv js ion o fNuc le ar Medi cin e, Un iv er sity o fM ichigan , Ann A rbor , M ichigan ; tUn iv er si ty o f M ichigan -Dearbo rn ,Dea rborn, M ichigan ; tAmhe rs t, Mass achu se tts ; and @Depar tmen to fChem is tr y, R en ss ela er Poly te chnic I ns titu te ,T ro y, N ew Yo rk


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11

11C-TRB

SD rats o f bo th sexeswere used for whole-bodyb iod is tr ibu tiona nd d osim etry stu die s. R ats w ere a ne sth etiz ed w ith e th yl e th era nd [ C ]TRBas i nj ec te d in to th e f emo ra l v ei n a t dos es o f 150900 @@Cin 250 z1 of fo rmu la te d s ol utio n. T he app ro xima tein je cte d ma ss o fTRB wasbe twe en 0 .3 -6 nmol/kg. An ima ls we reallo wed to rec ov er from an esthe sia afte r tracer in jection an dnorma l a ctiv ity was r es umed unti l t he time o f s ac rif ic e. An ima lsw ere k ille d b y d ec ap ita tio n a t sp ec ific tim es a nd who le tiss ue swer e d is se cte d out, c ounte d f or Cn a NaI(T l) we ll coun te r, andwei gh ed . B ra in s wer e d is se ct ed in to th e f ollow ing r eg ions : s tr iatum, c ere bra l c orte x (e ntire ), c ere be llum, in s om e c as es p on sm ed ulla , a nd re st o f b ra in . T he c arc ass w as m ea su re d fo r ra dioactivity in a dose calibra tor . Resul ts were correc ted for radioactived ec ay . T he y w ere c alc ula te d in te rm s o f p erc en t o f in je cte d d os eper organ (% ID /organ), and percent of injected dose per gram ofwet tiss ue , n orm aliz ed to a h yp oth etic al a nim al b od y w eig ht o f 1k g (kg % ID /g), to c orre ct fo r in div idu al a nim al w eig ht d iffe ren ce sand a ll ow for c ross -spec ie s compari sons o fd is tr ibu ti on da ta . Then um ber o f an im als u se d in eac h tim e p oin t are in dicated in th ef igureor table wherethe data are presented.Resultsforeach datap oin t a re e xp re ss ed a s th e m ea n v alu e p lu s o r m in us th e s ta nd arddevi at ion . Ca rbon- l 1 -s copol amine r at s tud ie s were ca rr ied ou t a ts im ila r ma ss a nd r ad io ac ti vi ty dos es a nd conditi on s. B ra in [ C]T RB radioactivitydistr ibutionwasalsoexaminedin a groupof1 1 m ix ed -se x C 57BL /6 m ic e, 2 0 30 g.t 6 0 mm fo llowin g 5 0 100 1@Ci. v. ( ta il v ei n) d os es o f[ C ]TRB .In b locking s tud ies to demons tr ate the degree of muscarin icspeci fi ci ty o fTRB uptake i n var ious t is sues, an ima ls were i nj ec tedsubcut aneousl y w it h e it he r ()QNB,2 mg/kg , ( )s copol amine ,8 mg/kg ,o r sa linevehic lea lone, 6090mm p rio r to tra ce rinjection.RatTissueMetabol i teAssayC arbon-i l-T RB (5 m Ci)w as injected via the fem oral vein intoa 1 90 -g fema le SD ra t. B lo od s ample s (2 50 @ il) ere c olle cte d int ubes con ta in ing 10mg o f pot as si um f luor id ea t 10and 20 mmfollow ing tracer injection. A t 30 mm, the rat w as killed and athird blood sam ple (1 m l) w as collected. B rain, heart, sm allin te stin e a nd pancre as wer e qui ck ly r emoved, m in ced, a nd th enh om og en ized w ith 3 m l o f e th ano l co nta in in g 1 0% acetic a cid .Whole b lo od samp le s w ere sh ak en b rie fly w ith th re e v olume s o fth e s ame e th anol/a ce tic a cid e xt ra cta nt. E ach tis su e m ix tu re wasth en c en tr if ug ed , c ounte d, a nd th e s uper na ta nt e xtr ac t wa s s ep arated from th e cell d eb ris. T he cell d ebris w as sh ak en w ith 3 m lo fpur e e th anol, c en tr if ug ed , a nd th e s ec ond extr ac t r emoved andcomb in ed w ith t he f ir st. T he deb ris wa s extr ac te d onc e mo re , th eresidue and pooled extracts for each tissue w ere counted tod ete rm in e re co ve ry e ffic ie nc y, a nd th e e xtra ct w as re du ce d to< 0.5 m l t o ta l v o lum e o n a r o ta ry e v a po r at or .A l iq u o ts o f t h er es ult in g concentr ate we re a pp li ed to g la ss -b acked s ilic a th inla ye r c hroma to gra ph y p la te s (E . Merc k, 1 5 cm) a nd d ev elo pe dw ith a s olv en t m ix tu re c on ta in in g CH2C12:E t2O:E t,N : E tOH,10:10:1 :1 .T h is s ys tem c le ar ly s ep ara te s TRB (R@0 .65) a nd desmethyl TRB ([email protected]) .Chromatogram radioactivity was examinedw ith a B erthold linear analyzer. The experim ent w as repeatedwith a second rat in which only the brain, heart, and smallin tes tine were examined.MonkeyPETIm aging

Qualitative images of brain radioactivity distribution wereconduct ed in f ema le p ig ta il monkeys (Macaca nemis tr ina ) we ighin g 6 7g , ( n = 3 ) a nd a 15 -k g fema le b ab oo n (P ap io a nu bis) (n

S CHEME 1 . S t r u ct ure for [11C ]TRB.

d ev elo pm en t o f [ CJTRBs a ra dio ph arm ac eu tic al fo rm ap pin g c en tra l mAChRs in m an .METhODSChemistryN-DesmethylTRB and auth en tic TRB wer ep r ep ar ed by me thods describedpreviously(26,27). Carbon-l 1-labeledmethyl iodide w as p repa red from [ C ]C02y modif icat ion of publ ishedmethods(28 ). Carbon -l 1 -scopo lamine wasprepa red by reduct ive[Cjmethylationf norscopo laminewi th [C]formaldehyde(18).Carbon-l l-TRB was labeled by [ C ]me th yl i od id e N - [' C]meth ylatio n o f d esm ethy l T RB , u sing an ap paratu s sim ila r indesign to the one used for synthesis of [C]scopolamine18).Gaseous [ C]methylodid e was bubble d i nt o a r ea cti on s olu tio no f 1.5 mg o f desmethy lTRB and 5 @t lf d ii sop ropy le thy laminein 200 i ii o fd ime thylf ormam ide coo le d to 50n a c on ic al v ia l.T he reaction vial w as sealed and heated at 80 or 4 mm, andth en 6 00 @slf w ate r w ere a dd ed . T he d ilu te d re ac tio n s olu tio nw as p as se d th ro ug h a s ho rt s olid -p ha se e xtra ctio n c olumn o f C -18s il ic awhi ch r et ai ned the c rude produc t [ C]TRB.his columnwas then rinsed with water (2 x 1 ml) and dried for 1 mm by ahighpressureflowofhelium (10bar).The adsorbedcrude productw as eluted from the extraction colum n onto a 9.6 X 150 m mam ino /cy an o b on de d p hase 5 m icro n silica sem iprep arativ eHPLCcolumn(ESIndustries,M arlton,NJ)by a 3-mi/mmflowofchromatographic solvent (93:7 acetoni tr il e: i sopropanol) . Af ters ep ara tio n o f [ C ]TRBr ete ntio n time 7 mm) f rom exc es s p recur so r desme thylTRB (10 mm) was e ff ec ted, t he solvent wasremove d from th e [ C ]TRBrac ti on by rot ary evapora ti on andth e fin al p ro du ct w as fo rmula te d in is oto nic s alin e c on ta in in g10%ethano l.The chemica land radiochemica lpur it y, and spec if ic act iv it y o feach ba tch of[ ' C ]TRBas assayed by analyt icalH PL C usingseri esultraviolet(220 nm) and radioactivitylowdetectors. Column: 4.6 x 250 mm 5 im C-18; solvent: 3:1:1a ce to nitrile : m eth an ol: 2 0 mM pH 6 .7 KHPO4, 1 m l/m in ; k 'TRB1.8,k'desmethyiTRs.6.The l imit of massdetectionof thesetwoc ompo ne nts u nd er ro utin e c on ditio ns w as 0 .2 @ tg /m lo f f ormul at ed produc t sol ut ion . Typ ical speci fi ca ti ons for ba tches o f [ C]TRBwereasfollows:radiochemicalpur ity:>99% ;specificactivi ty a t end ofsyn thesi s: 0 .5 C i/@mol .The amoun t o f desme thy lT RB in thef inalpr oductrangedfr omnonedetectableo 20% ofth e m ass o f T RB presen t. T he tim e requ ired fo r sy nth esis andf ormu la tio n o f [ C ]TRBwas 3345mm, the end of syn thesi sy ie ld wa s 250320mC i ( 20 sA/ 20 mm ta rg et ir ra di atio n), a ndth e d ec ay -c or re cte d y ie ld b as ed on [ C]CO2as 50%70%.Rodent Distr ibut ionFemale Sprague Dawley(SD) rats weighing 175200wereused in the regional brain distribution studies. A separate set of

424 The Journalof NuclearMedicineol. 33 o. 3 arch 1992


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Tissuekg%Rat (n= 35)Mouse(nil)dose/g*2 mm20 mm 30 mm 30 mm 30 mm 60 mm90 mm60mStnatum0.370.050.62 0.0760.520.02 0.0610.0080.0640.002 0.710.0970.74 0.040 .326 .042Cortex0.41 0.07 50.7 2 .0430 .52 .04 0 .062 .0080 .071 .006 0 .61 .070.59 0 .0 61 0.2 62 .069(whole)Cerebellum0.23.050.17 0.015 0.1 0.009 0.0450.005 0.0510.004 0.084 0.0120.07 2 0.00 50 .025 .003Blood0.048 0.0010.026 0.005 0.02 0.001 0.019 0.002 0.027 0.002 0.022 0.0020.024 0.0020.015 .001Striatium/1.7 0.243 .69 0.25 5.4 0.52 1.34 0.06 1.26 0.17 8.49 0.4210.3 0.612.7 .2Cerebellum%lD/brain2.930.454.03 0 .3 1 3 .7 0 .4 7 0 .5 7 0 .0 53 0 .6 6 0 .0 06 3 .3 4 0 .2 73 .1 1 0.54.29 .06*

Data are 9iven as the meantandarddeviat ion.

= 2), using the TCC 4600A three-ring, five-slice PET camera witha reso lu t ionof 13mm FWHM. Animalswereanes the tizedwi thi nt ramuscu la r ke tamine (15 mg/kg), and xy lazi ne hydroch lo ri de(2 mg/kg) ,and they were intubatedto preventaspirationofs al ivary sec re ti ons. Ke tamine was r epeat ed as necessary t o ma intam ane sth es ia . H e ad s wer e pos iti on ed in an a cr ylic h ead hold erw ith a b ite b ar a nd V elc ro stra ps. A fte r a p re lim in ary [ 5 0]H 20s can fo r pos it io ni ng and t o a ss ure norma l b lood f low , [ C]TRB,1 -1 .5 mCi/k g (0 .9 -6 nmo l/k g), w as in je cte d in tr av en ou sly . K inetic data wereacquiredusingan 18frame acquisi tionsequencewhic h la ste d b etw ee n 6 5-1 20 mmnin d ura tio n. S tu die s ra n 1 20mm unle ss te rm in ate d b y e qu ipme nt o r a nim al d iffic ultie s Y enousbloodwassampledperiodicallyformetabolites(seebelow).A m ath em atical atten uation correction w as ap plied in im agereconstruction. R egions of interest w ere draw n around the striatum /f ronta l c or te x ( se pa ra tio n was indis tin ct) , tempo ra l c ort exand cerebellum . Ipsolateral regions w ere com pared w heneverposs ib le t o minimize t he e ff ec ts o fs ide- to -s ide head moti on .To examinetheeffectofpharmacologicallevelsof scopolamineon bra in [ C]TRBlevel sn t he baboon, 20 mCi of[ C ]TRBwasadm in is te re d in tr av enou sly i n a 5 -m l bolu s a t t he b eg inni ng o f a1 20 -mm sc an . A t 1 10 mm , sc op olamin e 2 00 g ig /k g ( 0.6 7 jsmo l/kg) ,wasadministeredintravenouslyand the baboon's heart ratewas mon ito re d. Tw en ty m in ute s a fte r th e m ild ta ch yc ard ia d ueto scopolaminehad subsided,a second 120-mmscanwas carriedou t us ingano ther 20 mCi injec tionof L iC]TRBi th the samespeci fi c ac ti vi ty a s t he f ir st dose .Pr imateBlOOdPlasmaMetabol fteAssayAn anesthe ti zed f emal e p ig ta il monkey we igh ing7 kg wasg iv en an in trav en ou s injectio n of 10 mCi o f [C ]T RB .Bloodsamp le s w ere c olle cte d in tu be s c on ta in in g 1 0 mg o f p ota ssiumflu orid e (to m in imm2@oag ula tio n) a t 5 , 1 0, 2 0, 3 0, 4 0 mm (5 00p 1), 60 an d 80 mm (1 m l), and cen trifu ged fo r 1mm . T he p lasm awas s ep ar ate d w ith a p ip et a nd app li ed to a C -18 Sep -Pak p re cond itio ne d w ith 5 m l o fm eth an ol fo llowe d b y 1 0m l o f 1%a qu eo usammo nium carb on ate. A 1 5-m l p ortion o f 1% aq ueou s ammon ium car bona te was p as se d t hr ough th e Sep -Pak a fte r t he p la smaan d th e aqu eo us e lue nt (solub le m etabo lite fractio n) w as co llected. T he S ep -P ak w as th en w ashe d w ith 15 m l o f eth an ol toelu te the LiC]TRBract ion . The red b lood cell pe llet , e luentf rac tions ,and Sep-Pakafter f inal r insewerecoun ted C.he

recovery efficiency for TRB of this assay was determined incontrol experim ents using prim ate w hole blood spiked w ith authentic [C]TRB.otal unmetabolized[C]TRBn p la smawascalculatedas the activi tyin the ethanol f ract iontimesa recoverycorrection factor of 1.03 to compensate for TRB irreversiblyb ou nd to th e S ep -P ak . T he a bility o f th is a ssa y to s ep ara te [ C]TRB and m etabolites w as checked using rat blood m etabolites,withthe assumptionmadethat monkeyand rat metaboli teswerethe same chrom atographically. Blood samples (5 and 60 m m)f rom two more monkey s tud ie s in t he s ame animal on d if fe rento cc asio ns w ere a ss ay ed b y th is p ro ce du re a nd th ese re su lts d idnot var y f rom the f ir st by more than 10%.RESULTSRodent Dist ribu tionThe regional brain concentrations of [C]TRBn ratsa nd m ic e a t v ario us tim e p oin ts u p to 9 0 mm pos tin je ctio nare shown in Table 1. Rat cerebellum concentrationspeaked within the first 2 mm and then declined. W holecortex and striatum values w ere stable or continued to riseg ra du ally o ve r th e 9 0-mn p erio d. B ra in re gio n-to -b lo odactivity ratios at 90 mm ranged from about 3 for cerebellum to 3 0 fo r s tria tum . The b ra in lo ca liz atio n o f[ CJTRBin m ice w as consistent w ith rat d ata, show in g sim ilarstriatum -to-cerebellum ratios and % ID /brain values. T hefull body rat [@ C]TRB radioactivity distribution (% ID/organ ) in a separate set ofrats is show n in T able 2. D ecayc orre cte d ra dio ac tiv ity in most tissu es p ea ke d w ith in th efirst 15 mm , w ith liver, lung and kid ney containing largeamounts of radioactivity early on. By 15 mm, the braincontained the highest total amount of activity after theliver and rem ained so thereon. About 80% of the totaladmini st er ed rad ioac tivi ty cou ld be accoun ted for in theorgans and carcass (includes intestinal contents but notu rin e) 90 mm after in je ctio n.The 3 0-mm upta ke o f [ C ]TRBn ra t tissu es u nd ercontrol conditions or with QNB or scopolam ine pretreatmen t a re shown in Tab le 1 and F igur e lA . S im ila r d eg re eso f uptake inh ib it ion were observed using e ithe r pre tr ea t

TABLE 1Region alBrainDistributionof [11CITRBn Rode nts

Synthes is and Eva lua tion o f [11JTRBu lho lland e t a l 425


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%ln jected doseperrgan2mmi mm45 mm90mBrain3.78 0.535.77 0.774.06 0.393.34 .16Heart1.40 0 .161.26 0.080.76 0.080.31 .07Kidney7.31 1 .113.18 0.562.27 0.720 .74 .19Uver14.6 2.719.72 0.859.09 1.346.71 .38Lung8.47 1.302.99 0.501 .83 0.320.41 .08Testes0.78 0.051.08 0.140.8 6 0.0430.87 .03Spleen1.03 0.510.80 0.120.38 0.0330.15 .05Ovary0.22 0 .0030 .09 .020.06 0.01 50.02 .005Carcass73.7 10.368.7 6.2568.0 .4667.14 .17Total110 12. 092 .4 5.6487.1 4.2879.5 .8*

Data are g iven as mean stan dar ddeviation. =5 6atsperdatapoint.

[CITRBTotalrad/Targetorganrad/mCl*50CiBrain0.04262.13Heartwall0.04322.16Kidneys0.03821.91Liver0.02121.06Lungs0.04952.48Ovaries0.03531.77Red

marrow0.01 10.58Spleen0.02451.23Testes0.00820.41Totalbody0.001 10.06

TABLE 2WholeBody Biodistnbutionof [11CJTRBn Rats*

men t d rug , w ith t he g rea test r educti ons seen in the s tr ia tumand cortex (86%88%)ol lowed by the ponsmedul la(82%)and cerebe llum (50%).M orethan 80% ofthe heartuptakeo f [ C ]TRBo uld b e b lo ck ed b y QNB (F ig . l ).Compar ison wi th [11C]ScopolamineRat t issue radioactivi ty leve ls 30 mm after tracer injecti on u nd er c on tro l c on ditio ns and fo llowin g b lo ck in g p retre atm en t w ith co ld NB are shown for [C]TRBand[ C]scopo laminen F igure 1 . Tracer uptake in t is suesc on taining m uscarin ic recep to rs w as su bstantially greaterin the case of [C]TRB.O ve ra ll c on tro l b ra in le ve ls o fLiC]TRBere two-foldgreaterthan [C]scopolaminet2 mm and ap pro xim ately six tim es g re ate r th an [ C]scopolam ine brain levels at 90 mm (n = 35 nim als/tim epoint: data not shown). The percent reduction of controlupta ke s een a fte r QNB p re tre atment w a s s im ila r w ith b othElC]TRBndDosimet ry EstimatesThe e xp ec te d absorbeddo se s to humans fo r a 50-mC idos e o f[' C ]TRBre s hown in T ab le 3 . T he d os e-lim itin go rg an was th e o va ry . Dos ime try d ata w ere c alc ula te d u sin gthe rat biodistribution data from Table 2 follow ing theMIRD formalism (29). The percentadminis tered dose per

TABLE 3Ca lculatedbso rbedoseEs tima tesotheAdultrom

o rg an v alu es w er e modif ie d to re fle ct th e d iffe re nt p ro po rtions of organ to total body mass in rat and man (30).Residence times were obtained by integration under theo rg an tim e v ersu s a ctiv ity c urv es, w ith th e e ffe ctiv e h alflife of [ C]T RBassum ed to be equal to the physical halflife o f' Co r tim es e xc ee din g th e la st d ata p oin t. R es id en cetim es w ere en tered into the M IRDOSE 2 program (31) forthe generation of absorbed doses to selec ted target organsp er u nit o f admin istered activ ity .Metabo li sm Expe rimen tsThin-layer chrom atographic (TLC) exam ination of ratbrain extracts found at least 97% of extractable brainra dio ac tiv ity to b e id en tic al to u nm eta bo liz ed TRB . T her ecover y e ff ic iency o f t he thr ee -s tep ext ract ion p rocedur ewith b ra in tis su e wa s 86% and 83% respe ctiv ely fo r tworats.Heartand pancreasextracts([email protected]%)ikewiseshowed a s in gle r ad ioac tiv e spe ci es c oin cid en t w ith auth en tic TRB , in dic atin g n o ra dio ac tiv e m eta bo lite a cc umula tio n in th es e tissu es e ith er. Sma ll in te stin e h omog en ates yielded the low est recovery (76% ) of extractibleradioactivity of the tissues examined, and in contrast tothe above tissues, the intestinal extractwas predominantly(75% and 90% in the two rats) a single highly polarm etabolite fraction w hich did not m igrate from the origin.R at w hole blood exam ined by the extraction-TL Cm ethod (90% 95% recovery) at 10 m m follow ing tracerin je ctio n sh ow ed 8 3% u nmeta bo liz ed TRB . T he b ala nc eo fp la te r ad io ac ti vi ty was h igh ly pol ar . The unmetabol izedfra ctio ns o f re co ve re d ra dio ac tiv ity in ra t b lo od a t 2 0 a nd30 mm were approxim ately 65% and 50% , respectively.The resu lt sof Sep-Pak analys is of sequential samples ofvenous p la sma from one monkey fol low ing [C]TRBreshow n in F igure 2. C ontrol experim ents found 56% ofspiked authen ti c [ C]TRBn whole b lood part it ioned intothe plasm a a fter centrifugation. T he percentage of to talblood radioactivity (TRB plus metabolites) that partitio ned in to p la sma upon c en trifu ga tio n wa s in va ria nt o ve rth e se ve n tim e p oin ts a ssa ye d, w ith a m ea n p artitio n v alu eof55% 2% . A bout 97% ofauthentic [ C]TRBn plasma

426 The Jou rna l o f Nucl ea r Med ic in eo l. 33 o. 3 March 1992


5/8

0.1

1 0 09 0 I -

2

6680

70605040302010

00 20 40 60 80mm pos t i nj ec ti on

0.2 0.3 0.4 0.5 0.6 0.7.0

FIGURE2. Fract ionof m onkey plasm ato ta l ra dio ac tiv itywhich i s unme tabo lized [11C]TRBat vari ous time po in ts fo llo wing tracer in jection. Results fromoneanimal.

low es t in th e c ere be llum (low er p ortio n o f le ft s lic e). S copolam ine pretreatm ent resulted in a large reduction ofLiCJTRBptake in cortexand s tr iatum relativeto that inthe cereb ellum. R ep resen tative tim e-activ ity cu rv es of v arious brain regions of the pigtail m onkey are show n inFigure 4. The ratio ofleft stnatum (receptor-rich) to cerebe ll um ( recep to r- poor ) t is su e r ad io ac ti vi ty measur ed f romregions of interest w as 3.54at 5080mm, and the lefttem poral cortex-to-cerebellum ratio over this tim e fram ewas app ro xima te ly 3 .DI SCUSSI ONT his stu dy h as e xamin ed th e p ote ntia l o f [ C]TRBs aPET receptor m apping a gent u sing standard anim alm odels. TRB was one ofa series ofcom pounds developed

PANCREASADIPOSE

+QNBU CO NTR OLLIVERBLOOD

CORTEXCEREBELLPONS-MED

HEARTPANCREAS

B.c -i 1 SCOPOLAMINE

0 +QNB. CONTROLADIPOSELIVER

BLOOD0.0 0.1 0.2 0.3 0.4Kg % Dose /g ram

0.5 0.6 0.7

F IGURE1 . Radioac tiv ityevelsnselectedattissuesncont ro l and QNB pretreatedcondit ions,30 mm followingint ravenousinjectionof[11C]TRB(A)or [11C]scopolamineB).Each data pointre pre sents th e mean s ta ndard dev ia tio n o f a t le as t th re eanimals.was re co ve re d in th e e th an ol fra ctio n b y th e S ep -P ak a ss ay .Au then ti c [ C]TRBctiv ity and labeled metabo li tes werew eakly associated to the red blood cells (RBC), and m ostRBC a ctiv ity co uld b e re co vered in a n add itio na l stepwhich involved resuspending cells in 1.5 ml of pH 6.7phosphate-buf fered sa line , r ecent ri fugat ion and co llec tiono f th e su pe rn ata nt. Wh en th is se co nd ste p w as c arrie d o uto n th e RBCs from th e 4 0-mm samp le , S ep -P ak a na ly sis o fthe resulting supernatant show ed the sam e ratio of m etaboli testo TRB as in the f irst supernatant.This indicatedthat selec tiv e partitio nin g of ra diolabeled sp ecies b etw eenRBCs and p la sma d id not o ccur s ignif ic an tly and thus itis reasonab le to assum e the m etabolite fractio n of [C]TRB measured in p la sma by thi s a ssay accurat elyr e fl ec tsthe true m etabolite fraction in w hole blood.MonkeyPETImagesA repre se nta tiv e la te image o f [ C]TRBistr ibution inthe baboon brain is shown in the top row of Figure 3.A ctiv ity w as h ig hest in areas an atomically co rresp ond in gto the striatum and cortex (center and right slices) and

( to p r ow ) o r fo ul owu ig ( bo ttom ) scopo lam ine p re tr ea tment. B ra inlevelsare ascend ingromleftto righ t.Thes ligh tasymmetryncor tic al a nd subco rt ic al a reas is most lik ely an a rt if ac t caused byhead movementor misalignment.42 7ynthe sisand Eva luationof [11]TR Bulhollandet al


6/8

120.Lift SIri.tun6frcix0

!1@jT ime (mm)L If i TS i npOra l

individ ual using a S iem ens-C TI 9 31 cam era, w ith g oodcount statistics at 60-90 mm . T hus, sophisticated testre te st h uman stu die s a re p oss ib le w ith [ C]TRB .Muscar in ic Speci fic ityT he demonstration of in vivo specificity and selectivityfor binding sites is critically im portant in a potentialreceptor tracer. The regional brain and heart concentrations of [C]TR Bat tim es beyond 30 mm in rodentscorr elated well with known distributionsof mAChR (33,34) . Highest local izat ion occurred in the str iatum andcortex and the striatum-to-cerebellumr atioincreased in anearly linear fashion over a 90-m m period to values in thera ng e o f 1 0 fo r ra t, to 1 3 (mou se , 6 0 mm ), w ith th e in cre asebeingpr imarilydue to clearanceoftracer from cerebellum(Table 1) . The prominent local izat ion of [C]TRBnrodent brains at relatively short tim es is notew orthy in thatit com pa res very favo rably to , or exceeds that o f severallonger-lived isotope labeled ligandsstudied at substantiallylater tim e p oints, w here the o pportunity for clearance ofnonspecific activity w as greater. For instance, the rat caud ate[striatum]/cereb ellum ratios fo r [ 2 5I]QNBan d [3H]QNB at 4 hr are 7 (10) and 8 (35), respectively; for [125I]dexetim ide t he r at io in m ice at 120 m m is 10 (13), andt he r oden t r at io s f or [ 8 fl 2and 4 -f luo rodexe tim ides a t 60mm have been variou sly reported as being betw een 6 and25 (15 ,16 ).PET measurementsof brain regionsin pigtail monkeys(n = 3 ) s howed s tria tum (corte x)/c ere be llum ra tio s o f 3 4from 5080mm pos tin je ctio n. T hi s h ig h d eg re e o f lo ca lization, measured externally at 13 mm resolution in aprimate brain volume estimated to be less than 110 cm3(3 6), is a n en co ur a gin g in dica to r fo r th e u se o f [ C ]TRBin hum ans. It e xceeds by approx im ately tw o-fo ld the b ab oon s tria tum -to -c ere be ll um ra tio fo r L i C ]s co po lam in eunder similar scan conditions (Frey et al. , unpublishedresults),as well asthat of L 'C]benztropine1.5) measuredat 6.0 mm resolut ion at 60 mm (19) .M arked reductions of [C]T RBptake in muscariictis su es a fte r p re tre atm en t w ith p ha rm ac olo gic al a nta gonists (Table 1, Figs. l and 3) is evidence for m uscariicspec ificity an d saturab ility in the loc alization ofth is age nt.T he fact that the receptor-poorerebellum also showeda 5 0% b lo ck ad e (T ab le 1 , F ig lA ) a t first se em s c on fu sin g,until it is rem em bered tha t nearly all regions of the braincontain some mAC hR. Rat cerebellum containsabout 8%of the concentration of mAChR found in the striatum(34). A quick inspection of the blocked a nd contr ol 30-mm rat[C]TR Bata in T able 1 showsthat the reductionin rat cerebe llar [C ]T RB co ncentra tio n after block ad eam ounted to about 1 1% of the reduction in striatal [C]TRB concentration. The coincidence of these two measurementsis intriguing and seems to further suggest highi n v ivo speci fi ci ty f or [ CJTRB.An alternateexplanation for reduced tissue uptake following pretreatment is that poorly understood actions ofthe blocking drug unrelated to receptor occupation, such

FIGURE4. Representat ive t ime-tissueradioactivity curvesfor severalbrainreg io ns measured b yPET fol low ing int raven ous in jection of[11C]TRBn a pigtai lmonkey.originally as possible therapeutic agents for P arkinson'sdisease (26). It is a close chemical congener of QNB thatpenetr atesthe blood-br ain barr ierefficiently and possessesa high order of antimuscarinic potency (26) and receptoraffin ity . T he IC 50 v alu e w as 0 .7 nAt fo r (+ )-T RB m easu redv ersu s [3H]QNB b in din g to mou se b ra in m embra ne p re parations. This compares w ith IC50 values of 0.8n.M Tand 1.3 n.lI1for ()-QN Band scopolamine, respectiv ely , u nd er t he s ame te st c on ditio ns (2 5), a nd a re po rte dI C@for dexetimide of3.3 nM (13). T RB appearsto exhibits te reos el ec ti vi ty i n i ts ac ti on s s in ce the ( )somer o f TRBhas only about 10% of potency of the (+ ) isom er (26).The subtype specificity of TR B has not been studied, butit is likely to be similar to that of Q NB and scopolamine,which both show high affinity for all mAChR subtypes.U nlik e Q N B, T R B possesses an N -m ethyl gr oup thatmak es TRB fa irly e as y to la be l' C .Tissue Biodist ribut lon and Dosimet ryThe high [CJTRBrain levels seen in rodents (3% -4% of the injected dose/brain at 30 mm, Table 1) andprim ates (8% -l2% peak am ount, estim ated from PETdata) are notew orthy. In rats, [ C]TRBradioactivity levelsin the brain are 26times greater than those of [C]scopolamine at similar times (Fig. 2) and about four-foldh ighe r t han the r epor ted peak b ra in r ad io ac ti ve concen tr ations of [C]benztropine0 mm following injection intomic e (1 9). Robu st a ccumu la tio n o ftra ce r ra dio ac tiv ity inth e im ag in g ta rg et a re a is a n im po rta nt c ha ra cte ristic in aPET agent because it maximizes the imaging signal for agiven radiation dose. This is a particular concern withwhole-body PET cam eras which for geom etry reasons areless sensitive than narr ow diameter head-only units andtherefore requi re more act ivi ty in the f ield of view. Acoro lla ry is th at imagin g agents w ith h ig h tis su e a cc es s c angenerally be administered in lower amounts with betterdosimetry than agents with similar specificity but loweraccessibility to the target tissue. The absorbed dose estimate s fo r [ C ]TRB,a se d o n its ra t d is trib utio n, p erm ita cumulativeadult human dose of 50 mCi of[' CJTRBobe given. On the basis of studies presently underway inhum ans (32), it is estim ated that this quantity is sufficientto permit three 16 mCi [C]TRBtudies in the same428 TheJournalofNuclearMed icineol .33 o.3 arch1992


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as hem od ynam ic ch anges or stim ulation of tracer dispos itio n, m ig ht re du ce tra ce r d eliv ery to tissu e. T his p ossib ility c an b e e lim in ate d o nly a fte r c orre ctin g th e q ua lita tiv edata of these studie s by the true arterial input function.This was not done in the present work. However, thet im in g o f p re tre atments 3 090mm befo re tra ce r a dm inis tra tio n le sse ne d th e p oss ib ility th at b lo od flow a lte ra tio nsor o ther acu te e ff ec ts o f the b lock ing drug con tr ibu ted ina majo r way to th e o bserv ed d rama tic ch an ges in tra ceruptake betw een control and pretreatm ent studies.MetabolismLabeledmetabolites ofa PET radiotracermust be absentfrom the target tissue in ord er to a ccurately q uantify itst is su e d is tr ibut ion f ol low ing int ravenous inj ec ti on . Additionally, tracer m etabolites in blood should be m inim al,or well character izedthrough rapidanalyt ica l methods sothat correc tions can be made for radiolabe ledm etabol itesin a rterial blood. T his study has found that L C ]T RBsm etabolized at a steady rate in rat and m onkey, but nol abel ed me tabo li te s are pre sent in bra in t is sue. Furthermore, the larg e polarity difference b etw een T RB and itsla be le d me ta bo lite s p erm its ra pid and a ccura te a na ly sis o fu nm etab olized L ' C JT RBin b lo od b y sim ple solid -p haseext ract ion, and a s traigh forwardway of det ermin ing thetrue [CJTRBnput function in hum an studies is thusavailable.T he liver, lung and kidney appear to be organs formetabol ism and exc re ti on o f[ C]TRBin r at s, b as ed uponth ese o rg an s' sig nific an t ra dio ac tiv ity u pta ke a nd la ck o fuptake blockade by m uscariic antagonists. In fact, radioa ctiv ity le ve ls in liv er ro se sig nific an tly a fte r QNB (F ig .1A), possibly because of increased circulating [C]TRBaw ay from blocked tissues w hich norm ally w ould h aveserved as sinks for m uscariic tracer. The fact that nearly80% ofthe dose w as still present in the rat body at 90 mm(Tab le 2 ) sugges ts tha t, in thi s speci es a t l ea st , e xp irat ionof the label as m etabo lic CO2s a m inor path. The wallo f t he sma ll in te stin e re ta in ed s ig nific an t c on centra tio nso f ra dio ac tiv ity (d ata n ot sh ow n) th at w ere n ot b lo ck ab lewith QNB. Although this organ is known to containmAChR, analys is of intestinal t issue homogenates showedmost of the radioactivity prese nt here w as not L C ]T RBb ut in stead p olar o r no nex tractib le m etab olites.The ident it ie s o f the po lar me tabo li te s o f [ C]TRBnsm all in te stin e a nd b lo od a re u nc erta in a t th is tim e. T he irfa irly ra pid app ea ra nc e and hyd ro ph ilic c ha ra cte r s ug ge stsulfate or glucuro nide conju gate form atio n. A sim ilartransformation is prominent in the metabolism of scopolamin e in se ve ra l m ammal sp ec ie s (3 7). A sp ec ula tio n fo rth e n on ex tra ctib le in te stin al C-metabolites L'C]forma ld eh yd e, p ro du ced in th e liv er b y o xid ativ e N -d eme th yla tio n, th en se cre te d th ro ug h th e b ile d uc t to fin ally re ac tcovalently w ith proteins in the intestinal m ucosa. T heme thyl g roup in LN-'3C-me thyl ]ant ipy ri ne sha re s a s im i la rfa te in th e ra t (3 8), a nd th is ro ute may b e a common onefo r o th er C- labeled-methyl agents . Along these l ines,

moder at e l evel s o f nonex tr ac ti bl e i nt es ti na l r ad io ac ti vi tyhave been obser ved f ol low ing r at admin is tr at ion o f s ever alunrelated N-[' Cjmethylompounds (Muiho lland, unreported resu lt s) .Sy n t h e s i sA lthou gh the fa ct is seldom stated, a potential P ETtra ce r c an h av e th e o pp ortu nity fo r w id esp re ad u se o nly ifp ractical an d reliable m eth ods fo r its p rep aratio n are av ailable . [ C ]T RBs a sim ple, stable m ole cule that can bep roduced r ou ti ne ly by s tandar d L 'C]methy lodide me thylation in amounts greater than 250 mCi. In this respect,LiC]TRBoldsa clearadvantageverL'C]scopolamineand, with the poss ib le exception of LClbenztrop ine19 -21), it also appears sim pler to prepare than several otherreported pos it ron labeled centra l muscari ic t racers (14-16).CONCLUSIONST his s tu dy h as fo un d th at L ' C ]TRBa s fa vo ra ble u pta ke , d osim etry , m usc ariic sp ec ific ity a nd m eta bo lismcha ract er is ti cs i n animal s, s uggest ing a p romis ing mAChR

mapping agent in humans. A f inal con sidera tion , importa nt to the eventu al goal of single scan in vivo mAChRquantifica tio n, b ut b ey on d th e sco pe o f th is rep ort, iswhether L 'C]TRB-t issuepharmacokine tics are well -behavedo the extent they can adequate ly be described byk in etic mode ls u se d in PET fo r b indin g s ite s e stima tionunder nonequ il ib ri um s tudy cond it ions ( 10 ,22,23 ).Overall pharm acokinetics is a function of both thedrug's properties and the characteristics of the particulartissue/receptor system , and to a significant degree is specie s-v ariab le. T he d etailed d istrib utio n an d k in etic b eh avio r o f L C ]TRBn man con stitu te th e fin al s te ps in validation of this agent as a radiopharm aceutical for m uscar in ic r ecep to rs . Thes e invest ig at ion s a re unde rway and w il lb e sub ject s o f f utur e r epor ts .ACKNOWLEDGMENTSThe authors are grateful to Teresa Pisani and Dr. StevenPapadopoul is f or a ss is tance i n an ima l expe rimen ts , and to JamesMoskwa,RogerLunge r and John Carahe r f o r ope ra ti on o f t hec yc lo tro n. T ha nk s a re a ls o d ue to L in de r Mark ham fo r h er h elpin prep arin g th is m an uscrip t. T his w ork w as su ppo rted b y N ationalInstitutesofHealth grantsNS15655and NS24896-04,andDep ar tm en t o fE ne rg y g ra nt DE -F00 2- 87 ER60 52 8.

REFERENCES1 . Re in ik ain en K i, R ie kk in en P J ,Ha lo ne n T , L aa ksoM . De cr ea se dmuscar in ic r ec ep to r b in di ng i n c er eb ra l c or te x a nd h ip po camp us i n A lz he ime r'sdisease.LjfeSci 1987;41:453461.2 . B ow en DM , A lle n S J, B en to n i S, e t a l. B io ch em ic al a ss es sm en t o f s er ot in er gic a nd c ho lin er gic d ys fu nc tio n a nd c er eb ra l a tr op hy i n A lz he ime r' sdisease. JNeurochem 1983;41:266272.3 . Dan ie lsson E , EckernasS -A , Wes tl ind -Da the lsson A , e t a l. V IP- sensi ti veadeny la te cy cl as e, g uany la te cy cl as e, mu sca ri ni c r ec ep to rs , ch ol in e a ce ty lt ransferaseand acetyi cho l inest erase in b rain t is sueaff li ca t ed by Alzheimer 'sd ise ase /s en ile d em en tia o f th e A lz he im er ty pe . N eu ro bio lo gy o f A gin g429ynthesis and Evaluationof [11ITRBMulhollandet al


8/8

1988;9:153162.4 . L ange K W , W ell sF R , R ossor M N , i enn er P , M ar sd en C D . B r ai n mu scar in ic r ec ep to rs i n A lzhe ime r' san d Pa rk in son' sd is ea se . L ance! 1 989; 2: 1279 .5. Wh itehouseJ,TrifilettiRR,ionesBE ,eta l.Ne urotransm ittereceptora lt era ti ons in Hun ti ng t on 's d isease : aut o rad iog raphi c and homogena tes tu di es w it h s peci al r ef er en ce t o b en zodi az ep in e r ec ep to r comp lex es . AnnNeurol1985;18:202210.6 . M i zukawaK , OgawaN , So raYH , So ra I . A lt er at ionso f t he muscar in icc ho lin erg ic (mAC h) re ce pto rs in th e str ia tu m o f th e MPT P-in du ce d p ark in soni an mode l i n mi ce : i n v it ro quant it at iv e aut or ad io g raph ic al ana ly si s.Neuro sciet:l987; 81:10 5l0.

7 . D raye r B , ia szcza k R , C ole ma n E , et a l. M us carin ic ch olin ergic re ce pto rb in din g i n v iv o d ep ic ti on u si ng s in gl e p ho to n emi ss io n c omp ute d t omogr aphy and r ad io io di na ted qui nu ci di ny l b enZ il at e. JComputAs si st Tomogr1982;6:536543.8 . E cke lmanWC, RebaRC , Rzeszo ta rs kiW i , G ib son RE . Ex te rn al imagi ngofcereb ral muscar in i c ace ty l cho line recep to rs. Sc ience 1984;223 :291293.9 . E cke lmanWC, Eng R , Rzeszo ta rs kiW i, G ib son RE , F r anc is B , RebaRC .Use of 3-quinucidinyl4-iodobenzilateas a receptorbinding radiotracer.JNuc/Med 198526:637642.10. G ibson R E , W eck st ein D i, Jagoda E M , R zeszot ar sk i W i , R eba R C ,E ck elm an WC . T he c ha ra cte ristic s o fl-1 25 4 -I QNB a nd H -3 QNB in v iv oand in vitro.JNuclMed 1984;25:214222.1 1. H olm an B L, Gibson R E, H ill TC , Eckelm an W C, Albert M , Reba RC .Muscar in ic a ce ty lcho li ne r ec ep to rs i n a lzhe ime r' s d is ea se . I n v iv o imag in gw i th i od in e- 123 -l ab e led 3 -qui nu ci di ny l- 4- io dobenz il at e and emis si on t omography . JAMA 1985 ;254 :30633066 .12. PrenantC, Bane L, Crouzel C. Synthesisof [CJ-3-quinucid inylbenzila te(QNB). I Labelled Cmpd Radiopharm 1989,27:12571265.1 3. W ils on AA, Dan na ls RF, Rav en HT , F ro st ii, Wa gn er J r HN . S yn th es isand biological evaluation of [25j]@and [231]-4.iododexetimide, potentmuscarinic cholinergic receptor antagonist. J M ed Chem 1989;32:10571062.14. D annals R F, L angstr om B, R aver t H T , W ilson A A, W agner Jr H N .Synt he si s o f r ad io tr ac e rs f or s tu dy in g mu scar in ic cho li ne rg ic r ec ept or s i nthe living human brain usingpositron emission tomography: ICldexetimide and (qlevetimide.AppiRadiat Isot 1988;39:291295.1 5. W ils on AA, S ch eff el UA, Dan na ls RF, S ta th is M , Rav ert HT , Wa gn er J rHN. In vivobiodistr ibutionof two [18F)-labelledmuscarin iccholinergicreceptor uganda: 2-[18F J- and 4-(181 9-fluorodexe tim ide. L ife S ci1991;48:13851394.16. HwangD -R , DenceCS ,McK innon ZA , Ma th ia sC i, We lchM i . Posi tr onlabeledmuscarinicacetylcholinereceptorantagonist2- and 4-['59fluorod ex etimi de : s yn th es es a nd b io dis tr ib uti on . N uc i Med B io l 1 99 1 ;1 8:2 47 -252.1 7. V or a MM, F in n RD , Boo th TE. [N-me th yl - C] Sc op olam in e: s yn th es isa nd distribu tion in th e rat brain . J Labelled Compd Radioph arm1983;20:12291234.18. M ulholland OK, Jewett DM , Toorongian SA. Routine synthesisof N-[Cmethyljscopolamine by phosphite mediated reductive methylation with

[ Cj f o r ma l d e h y d e .p p i Ra d i a tI s o t 19 8 8 ; 3 9 : 3 7 3 - 3 7 9 .19. Dewey SL, M acGregor R, BrodieJD, et al. M apping muscarinic receptorsi n human and baboon b rain us ing [N- C-methyl ]- bennrop ine .Synapse1 9 90 ; 5 : 2 1 3- 2 2 3.20 . DeweySL, Brod ieJD, Fowle rJS , e t a l. Pos it ronemiss iontomography

(PET) studies of dopaminergic/cholinergic interactions in the baboonb r a i n . y n a p s e 1 99 0 ; 6 : 3 2 1 3 2 7 .21 . DeweySL ,Vo l kow ND, LoganJ , e t a l . Age- re la t eddecreasesn muscar in iccholinergic receptor binding in the hum an brain m easured with positronemis si on t omogr aphy (PET ) . I Neuros ci Re s 1990 ;2 7: 569575.22. FreyKA , Hichw aRD .Ehren kauferLE,AgranoffBW .Qu an titativ env iv o r ec ep to r b in di ng I II : tr ac er k in et ic mod el in g o f mus ca ri nic r ec ep to rbinding.'ivcailAcadSciUSA 1985;82:6716715.2 3. F re y KA, K oe pp eRA , Mu ih olla nd GK , e t a l. In v iv o mus ca rin ic c ho linergi c recep to r imaging in human brain wi th [Cjscopo lamineand posi tronemission tomography. CerebBlood Flow M ezab 199l;12:147154.24. Mu lh ol la nd GK , O tt o CA , Jewe tt DM , e t a L Radi osyn th es isand compariso ns in th e b io dis trib utio n o f c arb on -li m usc arin ic a nta go nists (+ )2 a-tr op an yl b en zi la te a nd N -me th yl- 4- pip er id yl b en zi la te [Ab st ra ctJ . J L abe/ledmpd Radiopharm198927:202-203.25. O tt o CA , Mu ih ol la nd OK , Pe rr ySE ,CombsR , ShermanPS , F ishe rS i. I nv it ro a nd c x v iv o e va lu ati on o f c yc li c am in oa lk yl b en zil ate s a s p ot en ti alemissiontomographyligandsfor the muscarin icreceptor .NuclMed Biol1989;16:5155.26 . A t ki nson ER, McRit chie DD, ShoerLF, e t a l . Pa rasympa tho ly t ic (an ti cholin erg ic ) e ste rs o f th e iso me ric 2 -tro pa no ls. 1 . G ly co la te s. J M ed C hem1977,20:161216l7.2 7. A rche r 5 , Bel lMR . U .S . Pat en t 3 ,1 45 ,2 10 , 1 964.28 . Crouze lC , Langs t romB. P ikeVW, CoenenHH. Recommenda tionsor ap ra ct ic al p ro du ct io n o f [ C ]met hy li od id e. A pp I R ad ia t I so t 1 98 7;3 8:6013.29 . Loev inger R, Be rman M. A rev isedscheme fo r ca lcu la ti ng abso rbeddosefrombiologicallydistributedradionucides.MIRD PamphletNo. 1,revised.New Yor k: S oc ie ty o fN uc le ar Medic in e; 1 97 6.30 . Roedler H. Accuracy o f i nt erna l doseca l cu la ti ons w i th spec ia l cons ide ration of radiopharmaceuticalsbiokinetics.RadiopharmaceuticalDosimetrySymposium, Oak Ridge National Laboratory, HH S-Publ (FDA) 8 1-8166;1980:120.3 1. Wa ts on E , S t ab in M , B oic h W . M IRDOSE2 . O ak R id ge A ss oc ia te dUn iv er si ti es , O ak R id ge , TN ; 1 98 8.32. F rey K A, K oeppe R A, M uiholland G K, K uhi D E. Q uantification ofr eg io na l c er eb ra l mus ca rin ic r ec ep to rs i n h uman b ra in w it h t he u se o f [ C-11]tropanylbenzilate and positron emission tomography [Abstract].JNuclMed 1990;31:885 .33.YamamuraHI ,SnyderSi. Muscarinicholinergicindingn ratbrain.P r o cNa tAc a dSc i USA 1974; 7 1: 1 725 1729.3 4. B ird sa ll N JM , H ulm e EC, B urg en A . T he c ha ra cte r o f th e m us ca rin icreceptors in different regions of the rat brain. P roc R S oc L and1 9 8 0 ; B 2 0 7 : 1 2 .35. Yamamu ra H I, Kuha r M i, Snyde rS i. I n v ivo i dent if ic at io n o f muscar in iccho li ne rg ic r ec ep to r b in di ng i n r at b ra in . B r ai n Res 1974; 80 :1 70 176.36. Pa rk erST. Whyigbrainsaresorare .n : Parke rST , G ibsonKR , eds.Language and inte ll ig ence i n monkeys and apes. Cambridg e: Un iv ers ityPress; 1990;129154.37 . Werne r VG, Schmid t K -H. ChemischeAna lysedesS to ffwechel svon ( )-S co po lamin b ei e in ig en S au ge tie re n. H op pe -S ey le r's Z P hy sio l C heml968;349:74l752.3 8. H ue tte r P , A lb ert K , B ay er E , Z elle r K P, H artm an n F . G en era tio n o fformaldehyde by N-demethylation of ant ipyr ine. Biochem Pharmacol1987;36:27292733.

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