A Potential Imaging Agent for SPECT Investigations of Benzodiazepine Receptors
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Radioiodinated 2'-.Iododiazepam: A Potential Imaging Agent for SPECT Investigations of Benzodiazepine Receptors Hideo Saji, Yasuhiko lida, Iwao Nakatsuka, Masaki Kataoka, Kazuhiko Ariyoshi, Yasuhiro Magata, Akira Yoshitake and Akira Yokoyama Faculty ofPharmaceutical Sciences, Kyoto Unwersisy, Kyoto, Japan and Environmental Health Science Laboratoiy, Swnitomo Chemical Company Ltd., Osaka@Japan relationshipshave shown that substitution with electron 2'-lododiazepam (2'-IDZ)isthe diazepam analogue iodinatedat withdrawinggroups such as halogens at position 7 and 2' the 2'-posilionof C-5 phen@1nngwh@hwas synthesizedand increases receptor affinity (2,17,18). lodination at position evaluated as a potential radiopharrnaceuticalfor invesligaling 7 yields 7-iodo-flunitrazepam, and the synthesis and bio brain benzodiazepine receptors by SPECT. The 1@l-2'-iododi distribution of this agent have been recently reported (13). azepam was SyntheniZedby halogen exchange reacOonand In this study, a diazepam derivative iodinated at the 2' pu@fledby HPLC.In vitrocompetitivebindingstudieswith3H- position, 2'-iododiazepam (2'-IDZ), was synthesized and diazepam,using rat cor@calsynaptosomalmembranes,showed the in vitro receptor binding affinityand biodistributionin that the affinity of 2'-IDZ for benzodiazepam receptors was higherthanthatin diazepamandflumazenil(R015-1788).Blo mice, including regional cerebral distribution, were stud distnbution studies in mice showed that the brain uptake of ied. The chemical structures of 2'-IDZ and related com 2'-iododiazepamwas rapidand profound,and inthe brainhigher pounds are shown in Figure 1. accumulationwas found in the cortexthan in other regions. Furthermore,the cortical uptake was displaced by benzodiaz MATERIALS AND METhODS epinergiccompounds.Invivouptakewasassessedby autora Sodium‘@I-iodide(specificactivity 81.4TBq/mmole)and 3H- diographicstudies.Thus, 2'-iododiazepamboundto benzoduaz diazepam (3.15 Thq/mmole) were purchased from Amersham In epine receptorsin vivo and therefore holds great potentialfor in temational Plc and Du Pont New England Research Products, v@,obenzodiazepinereceptorstudies. respectively.Diazepamand flumazenil(R015-1788)were a gen J NuciMed1993;34:932-937 erousgiftfromF. Hoffmann-LaRoche(Basel,Switzerland).Flu diazepamwas suppliedby SumitomoChemicalCo. Ltd. (Osaka, Japan).MaleWistarratsandddYmicewere suppliedby Japan SIC Co. Ltd. (Hamamatsu, Japan). enzodiazepines induce various pharmacological ef Radlolabellng fects which are thoughtto be mediatedvia benzodiazepine The radioiodinationof 2'-IDZwas accomplishedby halogen receptors in the central nervous system (1,2). Alterations in exchangereactionwith sodium‘@I-iodide.Eighty-fivemicro thedensityofbenzodiazepinereceptorshaverecentlybeen gramsof 2'-bromodiazepam(BDZ)was dissolvedin 107 @.ddim ethylformamide(DMF).Thissolutionwas addedto a mixtureof reportedin various disorders, such as epilepsy (3,4), Hun sodium‘@I-iodide(10 @d,37MBq),nonradioactivesodiumiodide tington's disease (5,6), hepatic encephalopathy (7) and Alz (0.8 @tgin0.25 @d0.1N NaOH),1-naphthalenesulfonicaciddihy heimer's disease (8). Visualization of benzodiazepine re drate(0.26 mg in 5 @lDMF) and copper (II) sulfate pentahydrate ceptorsin humanbrain hasthusbeenof greatinterest, and (0.26mgin 5 pi DMF) in a sealedvial. The reactionmixture was several “C-,75Br-, and ‘@I-labeledbenzodiazepines have heatedfor 1.5 hr at 100—105°C.Aftercooling,400 @lof 10% been developed for this purpose (9—16).The successful aqueoussodiumcarbonatesolutionwas addedto the reaction imaging of benzodiazepine receptors with these radiola mixture and the product was extracted with chloroform (2 x 0.5 beled compoundsand the superior radiation propertiesof ml).Thecombinedorganiclayerswereevaporatedunderastream ‘@Ifor SPEC!' promptedus to synthesize a radioiodinated ofnitrogen. The residue was dissolved in 100—150p1 of methanol, benzodiazepine analogue with high receptor affinity. applied to a reverse-phase, high-performance liquid chromatogra In the 1,4-benzodiazepine molecule, structure-activity phy (HPLC)column(LichrosorbRP-18,7.5 x 300 mm), and elutedwithmethanol:water:4-mMphosphatebuffer(pH7.4)(11: 5:4)at a flowrateof 1.5mi/rain(R@= 58miiifor 2'-BDZ, R@= 62 mmfor2'-IDZ).The fractioncorrespondingto 2'-IDZwas col ReceivedSept 10, 1992;revisionacceptedFeb.8, 1993. lected, evaporated to remove the residual organic solvent and Forcorrespondenceorreprintscon@ frJdraYokoya@PhD,Departmentof Raclopharmaceudcalchem@by,Facultyof PhaimaceuticalSdences, KyoloUrn sterilized by ifitration through a 0.22 pin Milex ifiter. @rernily,YoehklaSl*noadachi-cho,Sakyo.ku,Kyolo606-01,Japan. The radiochemicalpurityof the productwas determinedby 932 TheJournalofNudearMedicine•Vol.34•No.6•June1993 3H-diazepambindingversusinhibitor concentrationandwere the average of three to five experiments. MeasurementoftheOctanol/WaterPartitionCoefficient Thepartitioncoefficientfor2'-IDZwas determinedaccording to apreviouslyreportedmethod(21).A 20-idaliquotof radioio Cl dinated sample was mixed with 3 ml each of 1-octanol and 0.1 M phosphatebuffer(pH7.4)in a testtube.Thetubewasvortexed (3x 1 mm),incubatedfor 1 hr at roomtemperature,and then centrifugedfor 5 mm. The 5OO-@daliquots of each phase were removed and counted in a well-type Na! scintillation counter. CompoundRDiazepamHFludiazepamF2'-IDZI Blodlstrlbutlon StudIes Male ddY mice weighing about 30 g were administered ‘@I-2'- IDZ(18.5kBqin 0.1mlethanolicsalinesolution)throughthe tail vein. At thedesignatedtimesaftertheinjections,themicewere FIGURE1. chemIcalstruc turesof2'-IDZandrelatedcorn killed by decapitation and their organs were removed. For in vivo brain disthl,ution studies, the brains were dissected on an ice-cold plateaccordingto themethodof GlowinskiandIversen(22).All sampleswereweighed,andthe radioactivitywascountedusinga thin-layer chromatography (TLC) and analytical HPLC. The TLC well-typeNa! scintillationcounter.Resultswere presentedas the wasperformedonasilicagelplatewithachloroform:acetone(4:1) %dose/gtissueweight. solvent (R@= 0.62-0.68). HPLC was performed on a 7.5 x 300 Therelativebindingaffinityof ‘@I-2'-mZfor the benzodiaz mm Lichrosorb RP-18columnelutedwith methanokwater:4-mM epinereceptorwas determinedby usingknownagonistsandan phosphate buffer (pH 7.4) (11:5:4) at a flow rate of 1.5 mI/mm tagothsts with high affinity, which were injected into mice along (R1 = 62 mm). with 18.5 kBq of 1@I-2'-mZ. Flumazenil (1.0 mgfkg)was injected The nonradioactive 2'-IDZ and 2'-BDZ were synthesized from simultaneouslywiththe radioligand,but diazepam(10m@Jkg)and 2-iodo- or 2-bromobenzoic acid by addition of N-(4-chlorophe flunitrazepam (10 mg/kg) were injected intraperitoneally 30 min nyl)-N-methylethylenediamine, followed by cydlization and oxi before administration of the radioligand. The animals were sacri dation, according to the method previously reportedfor the cor ficed 1, 5, 20 and 60 mm after radioligandadministration,brain respondingfluoroanalogue,fludiazepam(19).Thestructureswere regionswere dissected,andtissueswere countedas described confirmed by infrared analysis (IR), proton nuclear magnetic res above.Theeffectof sodiumthiopentalon ‘@I-2'-mZuptakewas onance(NMR)andmassspectrometricanalysis.The detailsof also studied. Mice were injected intraperitoneally with sodium the synthesisandcharacterizationof these compoundswill be thiopental(100mg/kg)10 mm before radioligandadministration publishedelsewhere. andtreatedinthesamemanneras describedabove. To demonstratereceptorsaturation,18.5kBq of ‘@I-2'-mZ In Vitro BenzodlazeplneReceptorBIndIngStudIes was coinjectedwithvariousdoses of cold 2'-IDZ,rangingfrom The affinity of 2'-IDZ for benzodiazepine receptors was mea 0.01to 1.0mgfkg.Theanimalswere killed5 mmafterinjection sured by displacement of 3H-diazepam from a preparationof andthebrainsweredissectedandcountedas describedabove. synaptosomal membranes, according to the method of MOhler andOkada(20).Inbrief,thecerebralcortexfrommaleWistarrats Metabollsm (150-200g) was homogenizedin 20volumesof ice-cold0.32M Mice weighing about 30gwere injected intravenouslywith 55.5 sucrose using a Teflon-glass homogeneizer. The homogenate was kBq of 1@I-2'-IDZ.At designated times afterward, the mice were centrifuged at 1,000x g for 10 mm at 4°C.The supernatant was decapitated,and the brains were removedimmediatelyand ho recentrifuged at 20,000x g for 20 mm at 4°C. The pellet was mogenized in 1 ml of methanol. After centrifugation, the precipi resuspended in ice-cold 50 mM Krebs-Tris buffer (50 mM Tris tatewaswashedtwicewith 1 ml of methanol,andthewashes HQ buffer(pH7.4),118mMNaG, 4.8mMK@, 1.2mMCaCl2, were combined with the supernatant. The combined methanol 1.2 mM MgCl2)with polytron homogenizer, and dilutedwith the extracts were evaporated, and the resultingresidue was redis Krebs-Ths bufferto yield a synaptosomalmembranesuspension solved in a small volume of methanol and analyzed by HPLC on witha proteinconcentrationof 1.0mgprotein/mt. aLichrosorbP2-18column(7.5x 300mm)withmethanokwater: The bindingassays were performedby incubating1 ml of the 4-mM phosphate buffer (pH 7.4) (11:5:4) at a flow rate of 1.5 synaptosomal membrane suspension with 3H-diazepam (0.6 nM) mi/mm. anddifferentconcentrationsof competitorsin 1mlof Krebs-Tris buffer.Incubationswere performedfor 15 mmat 4°C,andthe AutoradlographicStudies sampleswere rapidly filtered througha WhatmanOF/B filter and Twogroupsofrats weighingabout250gwereused. Onegroup washedtwicewith5 ml of ice-coldassaybuffer.The