CLINICALSCIENCES

INVESTIGATIVE NUCLEAR MEDICINE

MyocardialImagingwitha RadioiodinatedNorepinephrineStorageAnalog

DonaldM.Wieland,LawrenceE.Brown,W.LosRogers,KathyC.Worthington,Jiann-longWu,NealH.Clinthorne, Charlotte A. Otto, Dennis P. Swanson,and William H. Beierwaltes

University of Michigan Medical Center, Ann Arbor, Michigan

Meta-lodobenzylguanldine(M-IBG), an iodlnatedaromatic analogof the hypo tensive drugguanethldine,localizesIn the heart of the rat, dog,and rhesusmon key. A comparativestudyof tissuedistributioninthe doghasbeenperformedwith five myocardiophlllcagents:thalllum-201, 1-125 16-lodohexadecanolcacid, H-3 norepinephrine,C-14 guanethidlneand 1-125M-IBG.The lasttwo compoundsgive heart concentrations and heart-to-blood concentration ratios similar to those of thalllum-201.PlanarandtomographicImagesof the heartsof the dogand rhesus monkeywere obtainedusing1-131or 1-123labeled M-IBG. Blockingstudieswith reserpinesuggestthat a majorcomponentof myocardlalretentionof M-IBG is Se questrationwithinthe noreplnephrinestoragevesiclesof the adrenergicnerves. The localizationof M-IBGinotherorganswithrichsympatheticinnervationandthe relative insensitivityof myocardialuptake to a wide range of loadingdoseslend additionalsupportfor a neuronalmodeof retention.

J Nucl Med 22: 22—31, 1981

Despite the widespread use of thallium-20l in myo norepinephrine cogener labeled with a radionuclide that cardial imaging, there remains a compelling need for an is generally available might be useful for routine myo alternative radiopharmaceut.ical (1,2). Recent attempts cardial imaging. Unfortunately, structure-activity-re to develop this alternative have focused on receptor lationship studies (12) suggest that little flexibility exists binding agents, such as radiolabeled /3-blockers (3,4) and for introducing a foreign atom other than fluorine (13) muscarinic antagonists (5); or substrate analogs, such into the norepinephrine molecule. It appears from studies as w-iodofatty acids (6,7). The latter compounds have with dopamine (14) that incorporation of radioiodine been used clinically, but high blood activity levels, due into the 6 position of the aromatic ring of norepinephrine to rapid metabolic release of radioiodide, have posed might provide a chemically stable derivative with a special imaging problems (8). biodistribution similar to that of norepinephrine, but a An approach to myocardial imaging—one that has multistep radiosynthetic scheme would be required. been for the most part neglected—can be based on the A plausible alternative to the use of a radiolabeled neurotransmitter norepinephrine. The heart is richly norepinephrine is the false neurotransmitter guanethi supplied by sympathetic nerves and its ability to rapidly dine (Fig. 1). This compound shares the same uptake, concentrate H-3 norepinephrine has been well docu storage, and release mechanisms as norepinephrine (15), mented (9,10). In 1976, Fowler and coworkers obtained but it is not metabolized by monoamine oxidase (MAO) scintiscans of the canine heart with C-I I norepinephrine or catechol-O-methyl transferase (COMT) (16). Car using a gamma camera (I I ). Thus norepinephrine or a bon-14 guanethidine (Fig. 1), when administered in pharmacological doses, selectively concentrates in the Received June 2, 1980; revision accepted Aug. 26, 1980. rat heart (17). Though guanethidine itself is not easily Forreprintscontact:DonaldM.Wieland,PhD,3685KresgeI,Univ. labeled with a gamma emitter, pharmacologically sim of Michigan Med. Ctr., Ann Arbor, MI 48109. ilar aromatic analogs of guanethidine, such as benzyl

22 THE JOURNAL OF NUCLEAR MEDICINE CLINICAL SCIENCES INVESTIGATIVENUCLEAR MEDICINE

NH purity. The radiochemical yield was 85-95%; specific activity was 1.1 mCi/mg. (/‘@@@\sss@I N.H I,,@,@@,CH@NH-C-NH2 Carbon-14 guanethidine sulfate. Radiochemical @ LS\%__/N-CH2CH2NH-@-NH2 purity was >95% as determined on silica gel, with n propanol:lO% NH4OH (3:1; Rf = 0.47) and n-buta FIG.1. Chemicalstructuresofguanethidine(denotespositionof nol:acetic acid:water (50:1 1:25; Rf = 0.17). The TLC C-14 label) and meta-Iodobenzylguanldlne(M-IBG). plate was visualized with Dragendorff reagent. Specific activity was 0.01 mCi/mg. Specific concentration in guanidine, can be labeled readily by incorporation of physiological saline was 50 zCi/cc. radioiodine into the phenyl ring (18). Iodine-I25 M-IBG. The radiosynthesis, by an iodide This paper presents an initial evaluation of radioio exchange technique, is similar to that previously reported dinated meta-iodobenzylguanidine (M-IBG) as a po for 1-125 para-iodobenzylguanidine (18). Synthetic tential radiopharmaceutical for imaging the myocar details will be published elsewhere. Recent improve dium. Specifically, we report: (a) tissue distributions of ments have lowered the radiosynthesis time to 2 hr. I- 125 M-IBG in the rat, dog, and rhesus monkey; (b) Specific activities of I-2 mCi/mg can be obtained rou comparative tissue distributions of C- 14 guanethidine, tinely in 90—98%radiochemical yield. Specific activities I-I25 M-IBG, H-3 norepinephrine, thallium-201, and up to 150 mCi/mg can be achieved in 40-50% yield. I- 125 16-iodohexadecanoic acid (I-HDA); and (c) Similar results were obtained in the syntheses of 1-123- imaging of the dog and rhesus monkey using 1-131- and and 1-131-labeled M-IBG. Radiochemical purity was I- 123-labeled M-IBG. >98% as determined by radio-TLC and radio-high pressure liquid chromatography (18). The final product was formulated in 0.9% bacterio MATERIALS AND METHODS static saline and the pH adjusted to 5—6by addition of Microanalyses were performed commercially. Thin pH 4 phosphate buffer. The specific concentration was layer chromatography (TLC) was done on precoated 0.2 mCi/cc. When stored in the dark at 4°C,I-I 25- and silica gel* or celluloset plastic strips. Tritiated norepi 1-131-tagged M-IBG showed <5% radiodecomposition nephrinet (25 mCi/mg), thallium-201,@ and sodium io in 3 wk. dide(I@125,1II-123,@and I@l3l*)wereobtainedfrom Tissue distribution studies. Dogs. Tissue distribution commercial sources. Proton magnetic resonance (PMR) studies were performed on female mongrel dogs (I 4-22 spectra were obtained on a spectrometer.** kg). Each animal received a single intravenous injection Synthesis of unlabeled compounds. The synthesis of of one of the following compounds: 100 sCi of Tl-20l, M-IBG. ‘/2H2S04has been described (18). The 16- 100 @iCiI-i 25 M-IBG, 100 sCi H-3 norepinephrine, I00 bromohexadecanoic acid, precursor to 1-125 iodohexa @zCiI-i 25 16-I-HDA, or 50 @sCiC- I4 guanethidine. The decanoic acid, was synthesized by the following method: dogs were killed at selected time intervals by intravenous I .08 g (4.0 mmole) of [email protected],tt injection ofsodium pentobarbital. For M-IBG, Tl-201, dissolved in 10 cc of 30% hydrobromic acid in glacial and I- 125 16-I-HDA, duplicate samples of 18 different acetic acid,*@was heated in a Parr pressure reaction tissues in each dog were weighed and counted in an au vessel for 6 hr at 100°C.After cooling to room temper togamma counter with corrections made for radioactive ature, the mixture was rotary-evaporated to dryness and decay, background, and counter efficiency. For H-3 the residue vacuum-distilled. The white solid, which norepinephrine and C-l4 guanethidine, duplicate sam distilled at 170—180°Cat 0.3 mm pressure, was recrys ples of the same tissues were weighed, oxidized in a tallized from 40—60°Cpetroleum ether to give 1.21 g of sample oxidizer,― then counted in a liquid scintillation white needles, m.p. 69.5—70.0°C; lit. m.p. 70—70.5°C counter with corrections made for background and (19). Yield was 91%. Anal.: calcd. for C16H31BrO2: C counter efficiency. Blood samples were obtained by 57.31,H 9.32,Br 23.83;found: C 57.60,H 9.32,Br cardiac puncture. To normalize for differences in animal 23.65; PMR (CDC13) 1.10—2.05[m,26,(Cj@)13], 2.35 weights, tissue concentrations are expressed as percent (m,2,Cjj@CO),3.40(t,2,Cf@Br),11.30(s,I,COOH). kilogram dose per gram (% kg-dose/g) (20). Synthesis of radiolabeled compounds. Iodine-I 25 Rats. Female Sprague—Dawleyrats weighing 200- 16-iodohexadecanoic acid. This radiosynthesis was 300 g were anesthetized with ether and injected with 25 based on the method of Robinson (6). A formulation of jzCi of 1-125 M-IBG by femoral vein. The rats were 9.4% ethanol, 2.3% polysorbate-80, and 88.3% physio anesthetized and killed by decapitation at selected time logical saline by volume was used. The specific concen intervals. Samples of heart, blood, liver, lung, and muscle tration was 0.2 mCi/ml. Radio-TLC on silica gel using were analyzed by the above procedures. n-hexane:ethyl ether:acetic acid (70:30:1 ; Rf = 0.25) and Monkeys. Following a restraining i.m. dose of ke on cellulose using n-hexane:ethyl ether:acetic acid tamine hydrochloride, monkeys were anesthetized with (320:80: 1) (Rf = 0.90) showed >98% radiochemical sodium pentobarbital and injected intravenously with

Volume 22, Number I 23 WIELAND, BROWN, ROGERS, WORTHINGTON, WU, CLINTHORNE, OTTO, SWANSON, AND BEIERWALTES

I00 zCi of I- 125 M-IBG. Three hours later, duplicate vehicle and then treated similarly. samples of 11 tissues were removed and analyzed by the Imaging. Tomographic imaging of the heart was above procedures. performed using a seven-pinhole collimator on a cam Blood curves. Blood curves were obtained in dogs era@1and a time-modulated coded aperture on a portable anesthetized with sodium pentobarbital. Venous samples camera11 (21,22). In each case, the heart was viewed in were obtained at seven selected time intervals and ana the LAO projection. lyzed, with results being expressed as % kg-dose/g. Specific-activity study. High-specific activity I-i 25 Analysis of radioactivity in the urine. Two dogs were M-IBG was synthesized by radioiodide exchange of 6.5 each injected with 1.0 mCi of I- 131 M-IBG and placed mCi of 1251with 25 zg of M-IBG . 1/2H2SO4in 0.20 in a metabolic cage. A 2-mi aliquot of the 24-hr urine ml of boiling water. Reaction time was 3.5 hr. Purifica collection was spiked with 5 mg of stable M-IBG. The tion (18) gave 2.8 mCi ofl-l25 M-IBG with a specific chemical form of the radioactivity in the urine was de activity of@―s130mCi/mg. Specific activities of 100, 50, termined by TLC on silica gel-G using ethyl acetate:95% 1.0, and 0.01 mCi/mg were obtained by addition of the ethanol (1:1; R@@fM-IBG 0.25; R1ofiodide = 0.76); requisite amounts ofstable M-IBG ‘1/2H2SO4to 500-zCi also in ethanol:28% NH4OH (3:1; RfOfM-IBG 0.14; aliquots of the stock solution. Concentrations were de R1of iodide = 0.85). The R1ofthe radioactive peaks(s) termined spectrophotometrically (Amax 228) using a was determined on a radiochromatogram scanner and standard curve. compared with the Rf of unlabeled compound as deter mined by fluorescent quenching under ultraviolet light. Control radiochromatograms were performed on 2-mI RESULTS nonradioactive urine samples spiked with I 5 zCi of I- Tissue distribution studies. Table 1 summarizes the 131 M-IBG, SMCiofsodiumiodide(I-I31),and 5mg distribution of 1-125 M-IBG in selected tissues of the rat, ofstable M-IBG. dog, and monkey. The monkey data are limited in scope Reserpine blocking studies. Two female mongrel dogs by the high cost of the animals. In all three species, were injected i.m. with I mg/kg reserpine followed by M-IBG showed selective localization in the heart. The a 100 tCi i.v. dose of 1-125 M-IBG 4 hr later. Two hours overall distribution patterns in the three species were after the M-IBG injection, the animals were killed and similar, with the major differences being: (a) more rapid tissue analyzed. Blood samples were taken by cardiac washout of radioactivity from the rat heart than from the puncture. The two female mongrel dogs that served as dog heart; (b) higher muscle concentrations (EM]) in the controls were injected with an equal volume of reserpine rat; and (c) increased liver uptake in the monkey. The

TABLEMONKEYSAnimalNo.1.DISTRiBUTIONOF M[1―IJI000BENZYLGUANIDINE(M-IBG) IN RATS,DOGS,AND of Animals TIme (hr) Heart BlOOd Liver Lung Muscle HIBt Tissueconcentration(% kgdose/g) Rats 6 0.5 1.09 0.03 0.36 0.69 0.25 36 (0.87—1.47) (0.03—0.04) (0.27—0.44) (0.44—1.03) (0.17—0.40) 5 1.0 0.68 0.04 0.27 0.38 0.14 17 (0.62—0.75) (0.03—0.04) (0.23—0.32) (0.31—0.42) (0.12—0.17) 5 4.0 0.19 0.02 0.06 0.11 0.10 10 (0. 14—0.23) (0.02—0.02) (0.03—0.08) (0.10—0.12) (0.08—0.15) Dogs 2 0.08 0.46 0.04 0.57 0.98 0.06 12 (0.43—0.49) (0.04—0.05) (0.52—0.62) (0.87—1.10) (0.05—0.07) 2 0.50 0.47 0.02 0.34 0.22 0.02 23 (0.44—0.50) (0.02—0.03) (0.29—0.39) (0.13—0.32) (0.02—0.03) 2 2.0 0.63 0.03 0.29 0.95 0.05 21 (0.60—0.67) (0.03—0.03) (0.26—0.32) (0.64-1.26) (0.04—0.06) 2 6.0 0.31 0.03 0.09 0.21 0.04 12 (0.28—0.34) (0.02—0.03) (0.09—0.09) (0.13—0.28) (0.04—0.05) Monkeys 3 3.0 0.64 0.02 0.76 0.17 0.02 32 (0.56—0.72) (0.02—0.02) (0.66—0.82) (0.16—0.18) (0.02—0.03)

. Mean and range. t Heart/blood. BlOOdsamples were taken by cardiac puncture. * Tissue data for other organs are available from authors on request.

24 THE JOURNAL OF NUCLEAR MEDICINE CLINICAL SCIENCES INVESTIGATIVENUCLEAR MEDICINE

DOGS*tAgentTABLE2. DISTRIBUTIONOFVARIOUS RADIOLABELEDHEARTAGENTSIN

dose/g)HeartBlood concentration (% kg Time (tw)Tissue LiverLungMuscle H/B@ 16[ 125l]iodo 0.08 0.58 0.07 0.26 0.28 0.12 8 hexadecanoic acid (0.53—0.62) (0.05—0.09) (0.25—0.27) (0.28—0.28) (0.11—0.13) 0.17 0.36 0.12 0.25 0.26 0.05 3 (0.32—0.40) (0.11—0.13) (0.25—0.25) (0.24—0.29) (0.03—0.07) 0.33 0.23 0.15 0.26 0.19 0.08 2 (0.17—0.29) (0.12—0.18) (0.22—0.31) (0.17—0.21) (0.07—0.09) Thalllum-201 0.5 0.38 0.01 0.20 0.29 0.05 38 (0.35—0.41) (0.01—0.01) (0.12—0.27) (0.21—0.36) (0.04—0.06) 2.0 0.37 0.01 0.14 0.15 0.08 37 (0.34—0.40) (0.01—0.01) (0.13—0.15) (0.13—0.17) (0.07—0.08) 4.0 0.21 0.01 0.22 0.10 0.10 21 (0.16—0.26) (0.01—0.01) (0.09—0.35) (0.08—0.11) (0.08—0.12) C-14 guanethidine 0.5 0.44 0.03 0.46 0.46 0.04 15 (0.43—0.44) (0.02—0.04) (0.33—0.58) (0.37—0.58) (0.03—0.04) 2.0 0.46 0.02 0.30 0.31 0.05 23 (0.41—0.50) (0.01—0.02) (0.29—0.31) (0.26—0.36) (0.04—0.05) 6.0 0.39 0.01 0.27 0.34 0.03 39 (0.31—0.46) (0.00—0.02) (0.24—0.31) (0.26—0.42) (0.03—0.03) M[125l]iodobenzyl 0.5 0.47 0.02 0.34 0.22 0.02 23 guanidine (0.44—0.50) (0.02—0.03) (0.29—0.39) (0.13—0.32) (0.02—0.03) 2.0 0.63 0.03 0.29 0.95 0.05 21 (0.60—0.67) (0.03—0.03) (0.26—0.32) (0.64—1.26)(0.04—0.06) 6.0 0.31 0.03 0.09 0.21 0.04 10 (0.28—0.34) (0.02—0.03) (0.09—0.09) (0.13—0.28) (0.04—0.05) 143 Norepinephrlne 0.5 0.74 0.04 0.27 0.18 0.07 18 (0.69—0.78) (0.04—0.04) (0.26—0.27) (0.12—0.23) (0.06—0.08) 2.0 0.39 0.03 0.17 0.07 0.04 13 (0.36—0.44) (0.03—0.03) (0.12—0.22) (0.05—0.08) (0.03—0.04) 6.0 0.21 0.02 0.27 0.11 0.06 10 (0.17—0.25) (0.02—0.03) (0.14—0.40) (0.05—0.18) (0.02—0.09)

C Tissue data for other organs are available from authors on request. t Two dogs per time Interval. t Heart/blood. Blood samples taken by cardiac puncture.

most encouraging features of Table 1 are the high heart 4. I-HDA has lower [H]/[B] and shorter myocardial concentration ([H]) and the heart-to-blood radioactivity tI,2 than the other agents. ratio ([H]/[B]) obtained in the monkey at 3 hr after A comparison of the time-activity curves of I- 125 injection. M-IBG, C-l4 guanethidine, and H-3 norepinephrine for Table 2 compares the tissue distribution of 1-125 the heart and blood of the dog are shown in Figs. 2 and M-IBG in the dog with those ofC-14 guanethidine and 3, respectively. The three compounds show rapid uptake H-3 norepinephrine. Data for Tl-201 and 1-125 HDA in the heart and similar efflux patterns up to 24 hr. Al are included for comparison. The major observations though the compounds show their highest [H] at early are: intervals, the exact time of maximum [H] varies slightly 1. Except for differences in lung activity, I-125-M- among the three agents. All three compounds are rapidly IBG and C-14-guanethidine show generally similar cleared from the blood; H-3 norepinephrine consistently tissue distribution profiles at 0.5 and 2.0 hours. gives the highest blood values at all time points from 2. H-3 norepinephrine showed the highest initial [H] 5-120 mm. of all the agents. Urinalysis study. Radio-TLC of the collected 24-hr 3. At 2 hr, M-IBG has slightly lower [H]/[B] and urines of two dogs administered 1-131 M-IBG showed [H]/[Lung] values than Tl-201 ; although the [H] and that >95% of the radioactivity was unchanged M [H]/[M] values of M-IBG are higher than those of IBG. Tl-20l at all time intervals. Specific-activity study. Figure 4 shows the concen

Volume 22, Number 1 25 WIELAND, BROWN, ROGERS. WORTHINGTON, WU, CLINTHORNE, OTTO, SWANSON, AND BEIERWALTES

.8 0 HEART 0 BLOOD @8 .7 2

3 4 TIMEO-IrS.) FIG. 2. TIme..activftycurves comparingthree agentsIn dog's myocardlum.EachpoIntrepresentsmeanof twodogs. -2 -I 0 I 2 LOG SPECIFIC ACTIVITY(mCi/mg) tration of 1-125 M-IBG in the heart and blood as a FiG. 4. ConcentratIon of I-125 M-IBG in dog's heart and blood as function of the specific activity of the administered functionof specificactivity.Eachpointrepresentsmeanoftwo dogs. compound. Within the 10,000-fold range of specific Animals were killed 5 mm after injection. BlOOdsampleswere ob activities evaluated, only the lowest specific activity (10 tamed by cardIac puncture. @zCi/mg)produced a change in the [H] and [B]; both values increased at this high loading-dose level. uptake of radioactivity in the left lung, suggesting the Imaging studies. Figures 5—8show representative presence oflung disease, a common occurrence in captive heart images obtained in the dog and monkey using ci primates (23). ther I-I23 M-IBG or I- I31 M-IBG. Standard as well as Reserpine blocking study. As shown in Table 3, pre tomographic images of the dog heart are included. Heart treatment of dogs with reserpine caused a 30% decrease images of consistent quality were obtained from 15 mm in themyocardialconcentrationof 1-125M-IBG. Blood to 3 hr in both species. The wall of the right ventricle can activity levels in the reserpine-treated dogs were higher be seen clearly in some of the tomographic slices. Using than the controls. 1-131 M-IBG, the monkey myocardium could still be imaged at 24 and 48 hr after injection with either a DISCUSSION pinhole or parallel-hole collimator. Of the four monkeys evaluated, only one failed to give All four chambers of the heart have a rich sympathetic a clear heart image. Use ofTl-201 in this same monkey also failed to image the heart. Subsequent injection of Tc-99m macroaggregated albumin showed decreased

I

I

TIME(Min.) FIG.5. Coded-aperturetomo@amsof normaldog'sheart(Dog1) Fig. 3. Tlme-actlvftycurvescomparingItree agentsIndog'sblood. obtained with 5 mCi m-[123ljlodobenzylguanldlne.Data were ob FIvedogswerekilledat eachtImepoInt.S.e.m.wastoo smallto tamed i Pwafter Injectlon,wfth imagIngtime 7 mm. Imageswere be shown. BlOOdwas obtained by venoussampling. reconstructed at 1-cm intervals, beginnIngat apex of heart.

26 THE JOURNAL OF NUCLEAR MEDICINE CLINICAL SCIENCES INVESTIGATIVENUCLEAR MEDICINE

FIG.6. Seven-pinholetomogramsofa normaldog'sheart(Dog1) @ ;v;;h 1 mCi of @n-[131l ::(A) with 5 mCi of m-[123l]iodobenzylguanidine.Datawere obtained0.5 monkey'sheart.(B)Parallel-holeimageof a normaldog'sheart.(C) hr after injection, with reconstructions beginning at apex of Pinhole-Imageof a normalmonkey'sheart.(0)Pinholeimageof a heart. normal dog's heart.

nerve supply, which serves as a link between the brain focusing on only a few, but nonetheless important, de and contracting heart muscles. Although 80% of the terminants of its in vivo distribution. norepinephrine present in the heart is synthesized there Should subsequent studies confirm that M-IBG re (10,24), the myocardium can avidly accumulate exog sembles guanethidine at both pharmacological and enous norepinephrine. The high initial concentrations tracer levels, the radiopharmaceutical evaluation of of H-3 norepinephrine observed in the dog heart (Table M-IBG becomes a straightforward task, since the dis 2) confirm the latter point. Studies have indicated that position and actions of guanethidine in the peripheral 50—70%of small intravenous doses of norepinephrine are sympathetic nervous system have been extensively rapidly metabolized by COMT and MAO (9). M-IBG studied (25—27).Guanethidine is both a neuron-blocking can thus be viewed as a “nonmetabolizable―norepi agent and a depletor of norepinephrine. Both actions, nephrine; it shares the same uptake, storage, and release though mechanistically different, require uptake of mechanisms as norepinephrine in adrenergic nerve ter guanethidine into sympathetic nerves. Blockade seem minals, but does not suffer the same metabolic fate (15). ingly entails binding to some cytoplasmic component of Use of radioiodinated M-IBG as a tracer could thus serve the neuron, whereas depletion involves uptake into the to simplify the complex disposition of norepinephrine by intraneuronal vesicles with subsequent release of nor

FIG. 7. Coded-aperturetomogramsof normalmonkey'sheart,obtainedwith 2 mCi m-[123l]lodobenzylguanldine. Data were obtained1 hr after injection,with Imagingtime20mm.Imageswererecon structed at 1-cm Intervals, beginning at apexof heart.

Volume 22, Number 1 27 WIELAND. BROWN, ROGERS, WORTHINGTON. WU, CLINTHORNE, OTTO, SWANSON, AND BEIERWALTES

loading doses oflabeled norepinephrine are injected (34). TABLE3.1-125M-IBG:RESERPINEBLOCKINGSTUDYIN Nonspecific binding at non-neuronal sites has also been dose/g)TImeDOGS(% kg observed with guanethidine (35), but apparently is a (tw)HeartBlood major factor only at pharmacological dose levels. 2. Long retention of M-IBG has been observed in the Control 2 0.46 0.03 (0.46—0.47) (0.03—0.03) spleen and adrenal medulla (18). The spleen has dense Reserpinet 2 0.31 0.05 sympathetic innervation, and the adrenal medulla is (0.25—0.37)(0.04—0.06) essentially a large sympathetic ganglion specialized for synthesis and storage of adrenergic hormones. . Blood samples were taken by cardiac puncture. 3. The tissue distribution of M-IBG (Table 2) re t I mg/kg I.m.; 4 1wlater 1-125 M-IBG given I.v. sembles that of C-l4 guanethidine. Thereisabundantevidencethattheneuronaluptake of norepinephrine is a saturable process that obeys Mi epinephrinestores.The dissociationof thetwomecha chaelis—Mentenkinetics (36). In vivo work with guinea nisms is best demonstrated by certain aralkylguanidines pigs has shown that saturation of heart uptake occurs that show predominantly one action or the other (28,29). with intravenous doses @l70pg/kg (10). Although From a radiopharmaceutical standpoint, recognition of M-IBG and norepinephrine do not necessarily have the these two pharmacological effects as distinct actions that same saturation levels for the carrier molecule, it is involve binding to separate loci within the adrenergic noteworthy that the routine loading dose of M-IBG was neuron is important: aralkylguanidines that exhibit both 10 zg/kg. In addition, the curve of specific activity actions should potentially show higher concentrations plotted against heart concentration shown in Fig. 4 is in the neurons and consequently higher uptake in sym strong evidence that the loading doses used in this study pathetically innervated organs, such as the heart. were well below the saturation level.The enhanced heart Structure-distribution relationship (SDR) studies now concentration observed at the highest loading dose in progress are focusing on radioiodinated aralkyl (specific activity = 10 zCi/mg) is consistent with the guanidines that possess this dual action. involvement of an extraneuronal uptake process. At this Guanethidine is thought to share the same neuronal high dose level, saturation of neuronal uptake has most uptake mechanism as norepinephrine. The uptake, often likely occurred, resulting in an incremental accumulation referred to in the older literature as uptake1 , is mediated of M-IBG in non-neuronal sites, such as the myocardial by an active transport process (30). This amine pump muscle cells (37). Thus an attractive feature of ra processes a variety of endogenous amines and basic dioiodinated M-IBG is that only at very low specific drugs. Both guanethidine and M-IBG are extremely activities is non-neuronal uptake in the heart observed. strong organic bases, with pKa's around I3.5 (31 ), so This obviates the synthetic difficulties and stability their neuronal uptake most likely occurs as the proton problems sometimes associated with agents requiring ated forms. The relative affinity of guanethidine for the high specific activity. Radioiodinated M-IBG with amine pump is one fourth that of norepinephrine (30). specific activities of 1—100mCi/mg can be attained in Note,however,thatrelativepumpaffinitywillnotbethe consistently high radiochemical yields by a rapid cx major determinant of overall neuronal accumulation of change technique. The use of radioiodinated M-IBG in a compound, but rather its affinity for intraneuronal this range of specific activities also provides a loading components, especially the storage vesicles. Reserpine dose well below the pharmacological levelsof substituted is generally thought to block selectively the vesicular benzylguanidines (38). uptake of norepinephrine in adrenergic neurons (32). In The rapid, high-yield radiosynthesis and in vitro sta addition, reserpine blockade of guanethidine uptake in bility of radioiodinatedM-IBG makeit a particularly the rat myocardium has been observed by Brodie and convenient agent. Metabolic studies are still in progress, coworkers (33). The results of the reserpine blocking but in vivo deiodination of M-IBG, as indicated by the study (Table 3) indicate that a considerable portion of thyroid activity levels, is very low at all time intervals the myocardial accumulation of M-IBG is by entrap studied. The pronounced stability of M-IBG parallels ment within the neuronal storage vesicles. Other, more the high thermodynamic stability of meta-substituted indirect clues for the neuronal uptake of M-IBG in the aromatics (39); the respective ortho and para isomers heart are: show considerably higher in vivo deiodination (18). 1. The efflux curve (Fig. 2) of M-IBG from the heart Radio-TLC of the urine of dogs administered 1-131 @ appears to be monophasic with t112 5 hr. If consider M-IBG has shown no major metabolites, but proof of the able extraneuronal (uptake2) or nonspecific binding had absence of metabolites—especially those arising from occurred in the heart, a biphasic efflux curve would have liver microsomal hydroxylation of the benzylic carbon been observed, the first component having a t112of 10—15 (40)—must await detailed analysis by high-pressure mm. Such a pattern has been obtained when large liquid chromatography.

28 THE JOURNAL OF NUCLEAR MEDICINE f.,I IMI(' A I Qr1L'@IeL'@ INVESTIGATIVENUCLEAR MEDICINE

The fact that M-IBG has a tissue distribution similar measure the array of physiological processes of the to that of guanethidine is in itself an encouraging finding, heart. demonstrating that large structural variations, including FUTURE WORK radioiodine incorporation, do not adversely alter the desired biodistribution. To date, studies reveal that the Attempts to document the noradrenaline-mimicking overwhelming determinant of heart specificity is the properties of M-IBG are continuing in hope of building guanidine group itself. a framework for its future use as a dynamic imaging The fatty-acid analog, 16-iodohexadecanoic acid, was agent. A comparison of the regional distribution of H-3 used in this study for comparison with the other myo norepinephrine and M-IBG in the heart should provide cardial agents. The unsaturated analog, 16-iodo-9- further evidence for the specific neuronal localization hexadecenoic acid, would be a more logical choice for of M-IBG. The change in M-IBG heart accumulation comparison since it has been more extensively studied, in response to various physiological and pharmacological but the synthetic precursor of the latter compound, interventions known to modify adrenergic nerve function I6-bromo-9-hexadecenoic acid, though commercially should also be instructive. An ongoing SDR study of available in crude form, is difficult to purify. The possible aralkylguanidines may produce a radioiodinated deny presence of both cis and trans isomers further compli ative with higher heart-to-liver and heart-to-lung con cates the purification. In contrast, 16-bromohexadeca centration ratios without sacrificing neuronal specificity. noic acid, the precursor of the 16-iodohexadecanoic acid, In addition, such studies could possibly lead to guanidine is conveniently synthesized in analytically pure form analogs amenable to rapid labeling with radionuclides, from the commercially available alcohol, juniperic acid. such as fluorine-18. Such an analog, if quantified by The I6-iodo-9-hexadecenoic acid and 16-iodohexadec positron-emission tomographic techniques, could po anoic acid have similar affinities for the myocardium tentially be used to measure noninvasively the kinetics (6). of storage and turnover in adrenergically innervated Functional aspects aside, an 1-123-labeled myocardial tissues in a variety of physiologic and pathophysiologic agent offers certain advantages over Tl-201 (t112 = 3 states. days). The shorter half-life of 1-123 (13.3 hr) and its 159 keV gammas, with 83% abundance, provide a higher FOOTNOTES photo flux per rad of absorbed dose at a desirable energy * Eastman Silica Gel I3 I8 1, Eastman Kodak Co., Rochester, level. In addition, the tissue attenuation coefficient for NY. the 70-90 keV photons ofTI-201 is 25%greater than for t Eastman Cellulose I 3255, Eastman Kodak Co., Rochester, the 159 keV gammas of 1-123. It should be noted that NY. * New England Nuclear, North Billerica, MA. I-i 23 has a 2.25% abundance of photons ranging from aUnionCarbide,Tuxedo,NY. 183 to 784 keV, which must be considered when selecting SCrocker Nuclear Laboratory, Davis, CA. collimators. It is also important to obtain 1-123 free of I CIBA—Geigy Corp., Ardsley, NY. the positron emitting 1-124 contaminant (41). ** Varian EM36OA. Although most of the images reported in this study tt AldrichChemical Co. were obtained by tomographic techniques, note that U Eastman Chemical Co. IIPackard306. standard pinhole images (Fig. 8) of the dog and monkey H Searle LFOV. hearts were of good quality. In fact the pinhole provides 1@Ohio Nuclear Series 420. high resolution and good discrimination against the high-energy gammas mentioned above. The higher liver ACKNOWLEDGMENTS uptake seen in the monkey, if also observed in humans, This work was supported by Grant No. CA-090I 5-02, Cancer Re might present difficulties for standard gamma imaging, search Training in Nuclear Medicine, from National Cancer Institute, but this should be minimized with tomographic tech DHEW; by ERDA Contract No. EY-76-S-02-2031;andby the Nu clear Medicine Research Fund. niques. The C-I4-guanethidine was provided through the efforts and The intent of this initial paper is not to suggest the use courtesy of Dr. M.F. Bartlett of the CIBA-GEIGY Corporation. of M-IBG as a myocardial perfusion agent but to stress The authors thank D. D. Marsh for technical assistance and Vicki its potential application to heart diseases characterized Van Wasshnova and Sally Synhaivsky for help in preparing the by alterations in sympathetic nerve function. The tissue manuscript. distribution data on M-IBG and other radiolabeled Aspecialthanksisextendedto Dr.J. A. McNamara,Jr., Dr. D.S. Carlson, and J. C. Ungerleider of the University of Michigan for do compounds in Table 2 should not be seen as a comparison nating the rhesus monkeys used in this study. of their potential for anatomical myocardial imaging. Several aspects of this work are the subject of a pending patent ap Thallium-201, 16-I-HDA and M-IBG are measuring plication from the University of Michigan. different physiological processes—most likely blood REFERENCES flow, metabolism, and adrenergic nerve activity, re spectively. No single radiopharmaceutical can serve to 1. MCKILLOP JH, MURRAY RG, TURNER JG, et al: Can the

Volume 22, Number 1 29 WIELAND, BROWN. ROGERS, WORTHINGTON, WU, CLINTHORNE, OTTO, SWANSON, AND BEIERWALTES

extent of coronary artery disease be predicted from thaI lene-carboniques de 8 a 2 1 atomes de carbone. Helv Chim lium-20l myocardial images? J Nuci Med 20: 715—719, Acta 12:463-492, 1929 I979 20. KIRSCHNER AS, ICE RD, BEIERWALTESWH: Reply. J 2. NIESS GS, LOGIC JR. RUSSELL RO, et al: Usefulness and NuciMed 16:248-249,1975 limitations of thallium-201 myocardial scintigraphy in de 21. VOGELRA, KIRCHD, LEFREEM, et al: A newmethodof lineating location and size of prior myocardial infarction. multiplanar emission tomography using a seven pinhole col Circulation59:1010-1019,1979 limator and an Anger scintillation camera. J Nucl Med 19: 3. JIANG VW, GIBSON RE, RZESZOTARSKI WJ, et al: Ra 648-654,1978 dioiodinated derivatives of fi adrenoceptor blockers for 22. ROGERSWL, KORAL KF, MAYANS R, et al: Coded-aper myocardial imaging. J Nuci Med 19: 9 18-924, 1978 tureimagingoftheheart.J NuclMed 21:371-378,1980 4. HANSON RN, HOLMAN BL, DAVIS MA: Synthesis and 23. T-W-FIENNES RN, LAPIN BA, DZHIKIDZE EK, et al: The biologicdistribution of radioiodinated $-adrenergic antago respiratory and alimentary systems. In Pathology of Simian nists.J Med Chem 21:830—833,1978 Primates. Part I, T-W-Fiennes RN, Ed., Basel,New York, 5. GIBsoN RE, ECKELMAN WC, VIERAS F, et al: The distri Karger, 1972, pp 671-710 bution of the muscarinic acetylcholine receptor antagonists, 24. K0PIN IJ, GORDON EK: Origin of norepinephrine in the quinuclidinylbenzilateand quinuclidinylbenzilatemethiodide heart. Nature I99: 1289, 1963 (letter to Editor) (both tritiated), in rat, guinea pig,and rabbit. J Nuc! Med 20: 25. CHANGCC,CHANGJC,SUCY: Studiesontheinteractions 865—870,1979 of guanethidine and bretylium with noradrenaline stores. Br 6. ROBINSONGD:Synthesisof ‘23l-16-iodo-9-hexadecenoicJ Pharmacol Chemother 30: 213-223, 1967 acid derivatives for use as myocardial perfusion imaging 26. ABBS ET, DODD MG: The relation between the adrenergic agents. mt J Appl Radiat Isot 28: 149-156, 1977 neurone-blocking and noradrenaline-depleting actions of some 7. MACHULLA HJ, STOCKLIN G, KUPFERNAGEL CH, et al: guanidine derivatives. Br J Pharmacol 51: 237-247, 1974 Comparativeevaluation of fatty acids labeled with C-I 1, 27. GIACHETTI A, HOLLENBECK RA: Extra-vesicular binding Cl-34m, Br-77 and 1-123 for metabolic studies of the myo of noradrenaline and guanethidine in the adrenergic neurones cardium: concisecommunication. J NucI Med I9: 298—302, of the rat heart: a proposed site of action of adrenergic neurone I978 blocking agents. Br J Pharmacol 58: 497-504, I976 8. VYSKA K, HOCK A, FREUNDLIEBC, et al: Myocardial 28. FIELDEN R, GREEN AL: A comparative study of the nor imaging and measurement of myocardial fatty acid metabo adrenaline-depleting and sympathetic-blocking actions of lism using w-1-123-heptadecanoic acid. J Nuci Med 20: 650, guanethidine and (—)-$-hydroxyphenethylguanidine. Br J 1979(abst) PharmacolChemother30:I55—165,1967 9. WHITBYLG,AXELRODJ,WEIL-MALHERBEH:Thefate 29. FIELDEN R, GREEN AL: Optical isomers of fi-hydroxy of H3-norepinephrine in animals. J Pharmacol Exp Ther 132: phenethylguanidine: effects on heart noradrenaline and on 193—201,1961 sympathetic blockade. Br J Pharmacol Chemother 32: 10. CROUTJR: The uptake and releaseof 3H-norepinephrineby 350-359,1968 the guinea pig heart in vivo.Arch Exp PáiholPharmacol 248: 30. MAXWELL RA, FERRISRM, BURCSUiE: Structural re 85-98,1964 quirements for inhibition of noradrenaline uptake by phen Ii. FOWLERiS, WOLF AP, CHRISTMANRD. et al: Carrier-free ethylamine derivatives, desipramine, cocaine, and other @ ‘C-labeledcatecholamines. In Radiopharmaceuticals, Su compounds. In The Mechanism ofNeuronal and Extraneu bramanian G, Rhodes BA, Cooper iT, SOddVi, Eds., New ronal Transport of Catecholamines. Paton DM, Ed., New York, Society of Nuclear Medicine, 1975,pp 196—204 York, Raven Press, I976, pp 95- I53 12. TRIGGLEDi: [email protected] Medicinal 31. DAVIS TL, ELDERFIELD RC: The determination of the Chemistry. 3rd ed. Part II, Burger A, Ed., New York, ionization constants of guanidine and some of its alkylated Wiley-Interscience, I970, pp 1235—1295 derivatives. J Am Chem Soc 54: 1499- I503, 1932 13. CANTACUZENED, KIRK KL, MCCULLOH DH, CtaI: Effect 32. CAMPOS HA, SHIDEMAN FE: Subcellular distribution of of fluorine subs$itution on the agonist specificity of norepi catecholamines in the dog heart. Effects of reserpine and nephrine.Science204:1217—1219,I979 norepinephrine administration. mt i Neuropharmacol 1: 14. FOwLER iS, MACGREGOR RR, WOLF AP: Radiophar 13-22,1962 maceuticals, I6. Halogenated dopamine analogs. Synthesis 33. BRODIEBB, CHANG CC, COSTAE: On the mechanismof and radiolabeling of 6-iododopamineand tissue distribution action of guanethidine and bretylium. Br J Pharmacol 25: studies in animals. J Med Chem 19: 356-360, 1976 171—178,1965 15. NICKERSON M, COLLIER B: Drugs inhibiting adrenergic 34. TRENDELENBURGU: The extraneuronal uptake and me nerves and structures innervated by them. In The Pharma tabolism of catecholamines in the heart. In The Mechanism cological Basis of Therapeutics. 5th ed.,Goodman LS, Gil of Nueronal and Extraneuronal Transport of Catechol man A, Eds., New York, Macmillan PublishingCo, 1975,pp amines. Paton DM, Ed., New York, Raven Press,1976,pp 553—564 259-280 16. ABRAMSON FB, FURSTCl, MCMARTIN C, et al: The iso 35. BOULLIN Di: Evidencefor intra-cellular guanethidinestores lation, identification and synthesis of two metabolites of in the rat heart revealed after removal of calcium ions. Br J guanethidine formed in pig and rabbit liver homogenates. PharmacolChemother32:I45-155,1968 BiochemJ I13:143-156,1969 36. IVERSEN LL: Uptake of circulating catecholaminesinto 17. SCHANKERLS, MORRISONAS: Physiologicaldisposition tissues. In Handbook ofPhysiology. Section 7: Endocrinol of guanethidine in the rat and its uptake by heart slices. mt ogy, Volume VI. Adrenal Gland. Blaschko H, Sayers G, Smith J Neuropharmacol 4: 27-39, 1965 AD, Eds.,Washington, DC, American PhysiologicalSociety, 18. WIELAND DM, WU iL, BROWN LE, Ct al: Radiolabeled 1975,pp 713—722 adrenergic neuron-blocking agents: Adrenomedullary imaging 37. IVERSENLL: The uptakeofcatechol aminesat high perfusion with EIJIIJ..iodobenzylguanidine. J Nuci Med 21: 349—353, concentrationsin the rat isolatedheart: a novelcatecholamine I980 uptakeprocess.BrJ Pharmacol25:18—33,1965 19. CHUIT P, HAUSSERJ; Sur les acides-alcoolspolymethy 38. SHORT JH, DARBY TD: Sympathetic nervoussystem

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blocking agents III. Derivatives of benzylguanidine. J Med dog and man. Drug Metabolism and Disposition 4: 269-275, Chem 10:833-840,1967 1976 . . 41. BRESLOW K BARNES J, MAK 5K, Ctal: Acceptance testing 39. BRAENDLIN HP, McBEE ET: Halogenation. In Friedel- of ‘23l-sodiumiodide solution produced by the ‘27I(p Sn) @ Crafts and relatedreactions, Vol. 111,Pt. 2, OIah GA, Ed., 23Xe‘231reaction. Proceedingsof2nd International Sym New York, lnterscience, 1964, pp 1517-I 593 posium on Radiopharmaceuticals. March 19-22 1979. 40. TURNBULL LB. TENG L, NEWMAN i, Ctal: Disposition of Seattle, Washington,New York Societyof Nuclear Medicine, bethanidine, N-benzyl-N',N―-dimethylguanidine, in the rat, pp 43-54

MIDEASTERNCHAPTER SOCIETYOFNUCLEARMEDICINE 11thANNUAL MEETING April 9-11, 1981 Unlformd Srvlces B.thesda, Maryland Unlvsrslty of H•althSrvlcs ANNOUNCEMENTAND CALLFORABSTRACTS TheScientific ProgramCommitteeof the MideasternChapterof the Societyof NuclearMedicinesolicits thesubmis slon ofabstractsfrom membersand nonmembers ofthe Socletyof Nuclear Medicineforthe 1Ith Annual Meeting tobe held April 9-11, 1981in Bethesda,Maryland.The programwill includesubmittedpapers,invItedspeakers,teaching sessions,andexhibits. Abstractsshouldnotexceed300wordsandshouldcontainastatementof purpose,themethodused,results,andcon clusions. The name of the author presenting the paper must be underlined. Originalabstractsandfourcopiesshouldbesentto: Charles D. Teates, M.D. Dlv.of MedicalImaging Dept.of Radiology Box 486 Univ. of Virginia Medical Center charlottesville, VA 22908 TheprogramwillbeapprovedforcredittowardtheAMAPhysician'sRecognitIonAwardunderContinuingMedical Education, Category 1,through the Society of Nuclear MedIcine. Forfurther information concerning the program,write or telephone (804)924-5201the ProgramChairmanlisteda- boveor E.U.Buddemeyer,Sc.D.at (301)528-6890. ABSTRACTSMUSTBERECEIVEDBYFEBRUARY1,IN1.

THE FIRST HIGH COUNTRY NUCLEARMEDICINE CONFERENCE RockyMountaInChapter MIssourIChapter March28-AprIl 4, 1981 Mark Hotel-Lionshead Vail, Colorado The FirstHighCountry NuclearMedicineConferencewill beheldatVailfrom March28-April 4,1981atthe MarkHotel at Lionshead. The program will feature discussionof advancedtopics in nuclear medicineand there will be time for skiing. Invited speakersinclude Drs. Daniel Pavel, John Keyes, Bill Klingensmith, and Robert Carretta. For further Informationcontact: ThomasRavin,M.D. Dept.of NuclearMedicine PenroseHospital 2215N.CascadeAve. ColoradoSprings,CO 80907 Tel: (303) 630-5242

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