MIBG describedherearenotlikelytocome intoplayinpatientsgiven diagnosticor commontherapeuticdoses of radioiodinatedMIBG.

KeyWords:metaiodobenzy@uankJine;tyramine;indirectsympathomimeticaction;neuronaluptake;vesK@ularmonoaminetransporter

J NucI Med 1999;40:1342—1351

ecausemetaiodobenzylguanidine(MIBG) is takenupby and concentrated in tumors originating from the sympathetic nervous system, radioiodinated MIBG is used widelyto diagnose and treat these disorders. Several aspects of thepharmacology of MIBG have been reviewed (1—3).MIBGacts as an alternative substrate of the neurona! norepinephrine transporter (neuronal uptake [uptake1], according toIversen [4]) and is therefore avidly taken up by postganglionic sympathetic neurons (5,6) and cells derived from theseneurons, including adrenomedullary chromaffin (7,8), human neuroblastoma (SK-N-SH) (9,10) and rat pheochromocytoma (PC-12) cells (10). After uptake1, MIBG is furtheraccumulated within transmitter storage vesicles because it isalso a substrate for the reserpine-sensitive vesicular monoamine transporter (11). Other membrane-associated aminetransporters may likewise contribute to the distribution ofMIBG in vivo. For example, the extraneuronal monoaminetransporter (uptake2, according to Iversen [4]) is responsiblefor MIBG uptake by non-neuronal cells in the rat heart (12),and MIBG uptake observed in endothelial cells of thepulmonary circulation (13) and blood platelets (14) ismediated by norepinephrine or serotonin transporters knownto operate in the plasma membrane of these cells. Asexpected from its chemical structure, MIBG does not appearto interact with a- or @3-adrenoceptors and is metabolizedneitherby monoamineoxidasenorby catechol-O-methyltransferase(15,16).

Other aspects of the pharmacology of MIBG are less welldescribed. For instance, despite the fact that indirectly actingsympathomimetic amines and MIBG have certain propertiesin common (e.g., both are good substrates for uptake1 andthe reserpine-sensitivevesicular monoamine transporter),thepossibilitythatMIBG releasesnorepinephnneandexertsindirect sympathomimetic effects has not been explored in

Becausenothing is knownabout whether metaiodobenzylguanidine (MIBG) has tyramine-like actions, the sympathomimeticeffectsofMIBGweredeterminedintheisolatedrabbitheartandcompared with those of tyramine. Methods: Spontaneouslybeating rabbit hearts were perfused with Tyrode's solution(Langendoriftechnique;37°C;26 mL/min),and the heart rate aswell as the norepinephrine and dopamine overflow into theperfusate was measured before and after doses of MIBG ortyramine (0.03—10pmol) given as bolus injections (100 pL) intothe aortic cannula. K,,@and V@ values for the neuronal uptake(uptake1)of125l-MIBGand14C-tyraminewereobtainedinhumanneuroblastoma(SK-N-SH)cells. The K@of MIBG for inhibitionofthe 3H-catecholamine uptake mediated by the vesicular monoamine transporter was determined in membrane vesicles obtamedfrombovinechromaffingranulesandcomparedwiththepreviouslyreportedK@valuefor tyraminedeterminedunderidentical experimental conditions. Results: By producing increases in heart rate and norepinephrine overflow, both cornpoundshaddose-dependentsympathomirneticeffectsin therabbitheart.MIBGwasmuchlesseffectivethantyramineinincreasingheart rate (maximumeffect59 versus 156 beats/mm)and norepinephrine overflow (maximum effect 35 versus 218pmoVg).Tyrammnealso caused increases in dopamineoverflow,whereas MIBG was a poor doparnine releaser. At a dose of10 pmol, the increasein heart rate lasted morethan 60 mmafterMIBGand about 20 mmafter tyramine injection.Accordingly,thenorepinephrineoverflowcaused by 10 pmol MIBGand tyraminedeclined with half-lives of 57.8 and 2.2 mm, respectively.Theeffectsof both drugs were drastically reducedin heartsexposedto2 pmovLdesiprarnine.Thekineticparameterscharacterizingthe saturationof neuronaluptakeby ‘25l-MIBGand 14C-tyrammnewere similar for the two compounds: Kmvalues of MIBG andtyraminewere 1.6 and 1.7 pmoVL,respectively,and V@ valuesof MIBG and tyramine were 43 and 37 pmol/mg protein/mm,respectively. However, in inhibiting the vesicular 3H-catecholamineuptake,MIBGwaseighttimeslesspotentthantyramine.Conclusion: MIBG is muchlesseffectivethantyramineas anindirectsympathomimeticagent.Thisisprobablya resultof itsrelativelylowaffinityforthevesicularmonoaminetransporterandexplains the relatively poor ability of the drug to mobilizenorepinephrine stored in synaptic vesicles. The long duration ofMIBG action results primarily from the drug not being metabolized by monoamine oxidase. The sympathomimeticeffects of

Received Jul. 7, 1998; revision accepted Feb. 2, 1999.Forcorrespondenceor reprintscontact:Karl-HeinzGraefe,MD,Department

of Pharmacology,Universityof Wurzburg,VersbacherStrasse9, D-97078Wurzburg, Germany.

1342 THE JouRNAL OF NUCLEAR MEDICINE •Vol. 40 •No. 8 •August 1999

Sympathomimetic Effects of MIBG:Comparison with TyramineKarl-Heinz Graefe, Franz Bossle, Reinhard WOlfel, Albrecht Burger, Maria Souladaki, Dirk Bier, Klaus Dutschka,Jamshid Farahati and Heinz Bönisch

Departments ofPharmacology and Nuclear Medicine, University of Wurzburg, WUrzburg; Department ofNuclear Medicine, UniversityofEssen, Essen; and Department ofPharmacology, University ofBonn, Bonn, Germany

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detail. Therefore, spontaneously beating, isolated, perfusedrabbit hearts were used to compare the abilities of MIBG andtyramine, the prototype of indirectly acting amines, toinducenorepinephrinereleaseandincreasesin heartrate.We also compared the saturation kinetics for MIBG andtyramine as substrates of uptake1 and as inhibitors of thereserpine-sensitive vesicular monoamine transporter.

MATERIALS AND METhODS

MaterialsMIBG was synthesized as described by Wieland et al. (17).

Theirmethodwasslightlymodified;MIBG wascrystallizedasacetate salt and not as sulfate salt. As determined by highperformance liquid chromatography (HPLC) and ultraviolet detection, the purity ofthe drug was >99%.

@I-MIBGwas synthesized by a Cu(I)-assisted, nonisotopicexchangemethod(18). Sodiumdisulfite wasaddedto 40—80MBq125! (Amersham, Braunschweig, Germany), and the solution evapo

rated to dryness. Meta-bromobenzylguanidine (purity > 99%) andCuCl, both dissolved in acetic acid, were added and the mixtureheated to 180°Cfor 10 mm. Then, the acetic acid was evaporated,and the residue was dissolved in eluent and subjected to HPLC.Separation was achieved on a Purospher column RP-l8 (5 @tm;250 X 4 mm)(Merck,Darmstadt,Germany)with0.01moIILNaH2PO@/CH3CN(950/50; v/v) as eluent. The fraction containing‘@I-MIBGwas collected, the eluent evaporated and the productdissolved in isotonic phosphate buffer (pH 7.4). The specificactivity of 1251-MIBGwas determined to be 81.4 MBq/pmol.

Other drugs used in the study were desipramine hydrochloride,dopamine hydrochloride, (-)-norepinephrine bitartrate, nisoxetinehydrochloride, pargyline hydrochloride, reserpine, tyraminehydrochloride (Sigma, Deisenhofen, Germany), 3',4'-dihydroxy2-methylpropiophenone (Upjohn, Kalamazoo, MI), heparin (Liquerain; Hoffmann-La Roche AG, Grenzach-Wyhlen, Germany),3H-(nng-2,5,6)-(-)-norepinephrine (specific activity 2024 GBq/mmol), “C-tyramine(specific activity 1672 GBq/mmol) (NENDuPont, Dreieich, Germany) and Eagle's minimum essentialmedium, fetal calf serum (Gibco-Life Technologies, Karlsruhe,Germany).

Stock solutions of MIBG acetate (100 mmol/L) and tyraminehydrochloride (300 mmol/L) were prepared in deionized water. Onthe day of the experiment, the dilutions were made in saline.

Isolated Perfused Rabbit HeartsRabbits (1.8—2.3kg) of either sex and mixed strains were used.

After intravenous administration of heparin (500 U) and anoverdose of sodium pentoparbitone (80 mg/kg), they were killed byexsanguination. Hearts were quickly removed, the aorta cannulatedand the coronary vessels perfused (Langendorif technique) withmodified 1@'rode's solution of the following composition: 118mmol/L NaCl, 4.5 mmol/L KC1,25 mmol/L NaHCO3, 1.4 mmolfLNaH2PO4, I .2 minol/L MgSO4, 1.4 mmol/L CaCl2 and I I mmol/Lglucose. The perfusion medium was maintained at 37°Candsaturated with 5% CO2 in 02 (resulting in a pH of 7.4); a rollerpump was used to deliver it at a constant rate of 26 mL/min. Thecoronary perfusion pressure and the left ventricular pressure of thespontaneously beating heart were monitored with Isotec pressuretransducers (Hugo Sachs Elektronic, March-Hugstetten, Germany)connected to a side arm of the aortic cannula and a saline-filled,latex balloon inserted into the left ventricle, respectively. The heartrate and the left ventricular pressure amplitude were derived from

the left ventricular pressure signal and recorded by a computerassisted signal processing system (MacLab system and Chartprogram; ADlnstruments, Castle Hill, New South Wales, Austraha). After an equilibration period of 30 mm, MIBG, tyramine orvehicle (solvent of the drugs) was given as a bolus injection (100IlL) into the aorticcannula.In some experiments,the uptake1inhibitor desipramine (2 @imoWL)was present in the perfusionmedium throughout.

Three groups of experiments were performed. Group I served todetermine dose-response curves for the heart-rate-increasing aswell as the norepinephrine- and dopamine-releasing effects ofMIBG and tyramine. To this end, MIBG or tyramine doses of 0.03,0.1, 0.3, 1, 3 and 10 jimolwereadministeredin thatorderconsecutively at intervals of 10—35mm, and heart rate and leftventricular pulse pressure (LVPP), a measure of contractility, weremonitored. The heart rate response to MIBG or tyramine wasdefined as the increasein heart rate above the baselinevalueobserved before the injection of the first drug dose. To determinethe overflow of endogenous norepinephrine and dopamine into theperfusion medium, the venous effluent from the heart was collectedcontinuously at 5-mm intervals and analyzed for catecholamines.After injection of MIBG or tyramine doses of 0.03, 0.1, 0.3, 1, 3and 10 pmol, the number of samples collected at 5-rain intervalswas 2, 3, 4, 5, 7 and 9, respectively. A 5-mm collection periodbefore the first dose was used to obtain baseline values. Thenorepinephrine and dopamine overflow observed after MIBG ortyramine injection was corrected for the spontaneous (baseline)overflow. Because the solubility of MIBG was limited (concentration in stock solution 100 mmol/L), the highest MIBG (andtyramine) dose used was 10 @imol(i.e., 100 @.tLMIBG stocksolution).

Group II served to determine effects of a single dose of 10 @imolMIBG or tyramine both in the absence and presence of 2 pmol/Ldesipramine. After drug administration, heart rate and catecholamine overflow were monitored in these experiments for 60 mm.Group III experiments were used to study the effects of vehiclesolutions (i.e., 100 pL distilled water, saline or 100 mmol/L sodiumacetate) on heart rate and LVPP. The effect of 100 pL of 100mmol/L sodium acetate on LVPP was also determined in heartspaced at 300 beats/mn (field stimulation at a voltage of 9 V withsquare pulses of 3-ms width delivered at a frequency of 5 Hz).

For chemical analysis of norepinephrine and dopamine, lO-mLportions of the perfusate samples were first mixed (rotatory mixer,5 mm) with 1 ng dihydroxybenzylamine (internal standard), 500 @tLTris[hydroxymethyljaminomethane (TRIS)-HCI buffer (2 molIL,pH 8.7) and 40 mg A12O3and then filtered (GF 52; Schleicher &Schuell, Dassel, Germany). The AI2O3remaining on the filter waswashed twice with 1 mL distilled water and then eluted twice with100 jiL HC1O4(0.1 mol/L), respectively. After pooling of the twoeluate fractions, 100 @iLof the eluate was subjected to HPLC. TheHPLC system consisted of a type 515 pump, an automatic sampleinjector WISP 717 (Waters, Eschborn, Germany) and a CoulochemH electrochemical detector connected to a triple electrode system(cell models 5021 and 501 1; ESA, Bedford, MA). The data weresampled and processed by the Millennium 2010 ChromatographyManager (Waters). For the chromatographic separation of norepinephrine and dopamine, a 5-pm Nucleosil 100 C18column (125 X3 mm internal diameter; Macherey-Nagel, Düren,Germany),maintained at 25°C,was used. The mobile phase (50 mmol/L.K@H2P04,0.1 mmol/L ethylenechaminetetraacetic acid (EDTA), 0.3mmol/L sodium octanesulfonate and 2% [v/v] methanol, pH 3.0)

SYMPAThOMIMETIC Em@crs OF MIBG •Graefe et a!. 1343

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was pumped at a flow rate of 0.8 mLlmin. The potential of the firstelectrodein theserieswassetto 350mV,thesecondto 300mV andthethirdto —350mV versustheinternalsolidstatepalladiumreference electrode. The mean 3,4-dihydroxybenzylamine hydrobromide (DHBA) recovery from the perfusate amounted to 72%; thismean value had an intra-assay coefficient of variation of 4% and aninterassay coefficient of variation of 7%. The results concerningnorepinephrine and dopamine were corrected for the DHBArecovery.

Human Neuroblastoma (SK-N-SH) CellsThe human neuroblastoma cell line SK-N-SH (subtype SY5Y;

American type Culture Collection, Rockville, MD) was used tocompare the saturation kinetics ofMIBG, tyramine and norepinephtine uptake mediated by uptake1. Cells were cultured in tissueculture dishes (60-mm diameter) with Eagle's minimum essentialmedium supplemented with 10% fetal calf serum. Initial rates ofuptake of ‘251-MIBG,‘@‘C-tyramineand 3H-norepinephrine weredetennined at 37°Cin N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfomc acid] (HEPES)-buffered Krebs-Ringer solution (KRS) ofthe following composition: 125 mmol/L NaCl, 4.8 mmol/L KC1,1.2 mmol/L Mg504, 1.2 mmol/L KH2PO4,1.3 mmoLfLCaCl2, 25mmol/L HEPES, 5.55 mmol/L glucose and 1.0 mmol/L ascorbicacid. Catechol-O-methyltransferase and monoamine oxidase wereinhibited by the presence of 10 @imol/L3',4'-dihydroxy-2-methylpropiophenone and 10 @imoWLpargyline, respectively. After1 mm of incubation with the labeled amines, cells were washedthree times with 2 mL ice-cold KRS and then solubilized in 1.5 mLof either 0.3 mollL NaOH (@I-MIBG) or 0.1% TritonXlOO(“C-tyramineand 3H-norepinephrine).The amount of radioactivityin 1 mL of solubilzed cells was determined in a gamma counter(‘251-MJBG)or a liquid scintillation counter (‘@‘C-tyramineand3H-norepinephrine). The protein content of the solubilized cellswas quantified by the method of Lowry et a!. (19). To increase thesubstrate concentration of @I-MIBG(0.1, 0.2, 0.5, 1.5, 5 and 10@.imol/L)and 3H-norepinephrine (0.03, 0.1, 0.3, 1, 3 and 10 @imol/L)in the incubation medium, the specific activities of the labeledamines were lowered by addition of unlabeled amines. On the otherhand, when the concentration of ‘4C-tyraminewas increased (0.5,1, 1.5,3, 6 and 10p.tmol/L),the specificactivityremained unchanged.

Initial rates of the uptake of @I-M1BG,@C-tymmineand 3H-norepinephrine were always determined in the absence (totaluptake) and presence (nonmediated uptake) of 10 ,.@mol/Lnisoxetine (a selective uptake1 blocker), and the saturable uptakemediated by uptake1 was calculated from the difference betweentotal and nonmediated uptake.

Membrane Vesicles of Bovine Chromaffin GranulesChromaffin granules were prepared from bovine adrenomedul

lary tissue by density gradient centrifugation over 1.6 mol/Lsucrose. Membrane vesicles were obtained after lysis of chromaffingranules (10 mm at 30°C,pH 6.0) in a medium containing 180mmol/Lurea,20 mmoIfLascorbicacid, 10mmol/L K2HPO@/KH2PO4and 0.5 mmol/L EDTA.After sedimentation (l44,000g for 20 mm),membrane vesicles were resuspended in 270 mmolIL sucrose/lOmmol/L TRIS-H25O4(pH 6.0) and stored at —70°C.One milliliterof this vesicle suspension contained approximately 1 mg proteinand 60—100nmol emdogenous epinephrime/morepinephrimewith atypical mixing ratio ofl:3. Before use, the pH ofthe freshly thawedvesicle suspension was adjusted to 7.3 with 1 mollL TRIS.

Uptake experiments were performed at a pH of 7.3 and at 30°C.The 5-mL incubation mixture contained 0.5 mL of the membrane

vesicle suspension as well as 330 mmolIL sucrose, 30 mmoIILTRIS-HC1 (pH 7.3) and 3 mmol/L adenosine triphosphate-MgSO4.After 10 mm of preincubation in the absence or presence of 0.1pmol/L reserpine, a selective inhibitor of the vesicular monoaminetransporter, and various concentrations of MIBG (3—1000pimol/L),the uptake was started by the addition of a 3H-catecholaminemixture (70% epinephrine plus 30% norepinephrine plus traceamounts of 3H-norepinephrine) to give final total catecholamineconcentrations of 13, 31 or 108 pmol/L. After 3 mm of incubation,a 4-mL aliquot of the incubation mixture was transferred into largeice-cold beakers containing 400 mmol/L sucrose and filteredthrough cellulose nitrate membrane filters (0.45 pm). After beingwashed with ice-cold sucrose, the filters were dried (80°C,30 mm),dissolved in scintillation cocktail and analyzed for radioactivity byliquid scintillation counting. A 0.5-mL aliquot of the incubationmixture was centrifuged (144,000g for 20 mm). The supernatantserved to determine the specific activity in the incubation medium(20,21),andthepelletwasusedforproteindetermination(19).

To measure the reserpine-sensitive uptake that reflects theuptake component mediated by the vesicular monoamine transporter, the vesicular 3H-catecholamine uptake was always determined in the absence and presence of 0. 1 pmol/L reserpine. Asshown previously for identical experimental conditions, the reserpine-sensitive vesicular 3H-catecholamine uptake proceeds at mitia.lrates for at least 3 mm, is saturable and exhibits (at a pH of 7.3)a Kmof approximately 10 @imol/Land a VmaxOf 10—20nmol/mgprotein/mn (20,21).

Data Analysis and StatisticsDose- and concentration-response curves for the various effects

of MIBG or tyramine were analyzed by fitting Hill's equation witha nonlinear least squares method to the experimental results. Hill'sequation of the form E = Em,,@D@@H/(EC50@@H+ D―@)@maximum effect; EC50,drug dose producing Emax/2nH, apparentHill coefficient = midpoint slope of the curve) was used to analyzedose-response curves showing increases in heart rate and catecholamine overflow (E) in response to the drug dose (D). Similarly, theequation used to analyze concentration-response curves relating thevesicular 3H-catecholamine uptake (U, expressed as percentage ofcontrol uptake) to the concentration ofMIBG (C) had the form UImax C@@/(IC5o@@H+ CnN) (I,,,@, maximum inhibition of uptake; IC50,

MIBG concentration producing@ K1values were calculatedfrom IC@ values, as described by Cheng and Prusoff (22). TheMichaelis-Menten equation was fitted to the saturation curvesrelating initial rates of uptake to substrate concentrations (i.e., tothe results obtained in SK-N-SH cells) by nonlinear regressionanalysis to give values of K,,,and V,,,,@.

Results presented are either arithmetic mean ±SEM or geomettic mean with 95% confidence limits; n is the number of expenments. The statistical evaluation of differences between means(Student t test; analysis of variance followed by the Bonferroni testfor multiple comparisons or by the test for linear trends betweencolumn means and column number) and the calculation of regression lines (y = a + bx) were performed according to conventionalprocedures. P values < 0.05 were taken to indicate statisticalsignificance.

RESULTS

Sympathomimetic Effects in Perfused Rabbit HeartIn spontaneously beating rabbit hearts, both MIBG and

tyramine elicited increases in heart rate. However, MIBG

1344 THE JOURNALOF NUCLEARMEDICINE •Vol. 40 •No. 8 •August 1999

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B Tyramine MIBG150 (1O@tmoI)

+100

CODE CODE

Tyramine

was much less effective than tyramine (Fig. lA). WhenHill's equation was fitted to the results by nonlinear regression analysis, the maximum response obtained by extrapolation was much less pronounced for MIBG (59 ±24 bpm)than for tyramine (156 ± 14 bpm; P < 0.01). Thepositive-chronotropic effects of 10 pmol tyramine andMIBG observed at the end of the dose-response experimentsof Figure lA did not differ from those observed in hearts inwhich 10 pmol tyramine or MIBG was the sole dose (Fig.lB). Figure lB also shows that the heart rate responses totyramine and MIBG were markedly reduced by the uptake1blockerdesipramine(2 pmol/L).

MIBG and tyramine also elicited a dose-dependent release of norepinephrine (Fig. 2A). But again, as for the heartrate response, the increase in norepinephrine overflowevoked by MIBG was much less pronounced than thatevoked by tyramine. The calculated maximum norepinephtine overflow induced by MIBG and tyramine was 35 ±8and 218 ±33 pmol/g (P < 0.01), respectively. The overflowresponse to both drugs was highly susceptible to inhibitionby desipramine (Fig. 2B). Qualitatively similar results wereobtained for the drug-induced overflow of dopamine (Figs.2C and D). Figure 2 also shows that 0.3 iimol tyramine and10 @imolMIBG were about equally effective not only inreleasing norepineprhine but also in releasing dopamine.Hence, the difference in the abilities of MIBG and tyramineto release dopamine appeared to be quantitative and notqualitative. The ratio of dopamine overflow to norepinephrine overflow induced by tyramine increased with increasingtyramine dose (0.037 ±0.015 at 0.3 @unoland 0.134 ±0.011 at 10 @.imoltyramine [n = 7 each; P < 0.01]). Thiskind of analysis was not possible with MIBG, because theincrease in dopamine overflow observed in response toMIBG was low and inconsistent.

MIBG and tyramine differed not only with regard to themagnitude of their sympathomimetic effects but also as tothe time course of their effects. As far as the heart rateresponsesto 0.03, 0.1, 0.3, 1, 3 and 10 jimol MIBG ortyramine are concerned (n = 7 each), the time to peakincreaseinheartratewas 0.2±0.1,0.4±0.1,0.6±0.1,1.9 ±Ungetragerte Radiosynthese von meta-[1231]- 0.4,3.6 ±0.6 and 8.6 ±1.0 mm, respectively, after administrationofMlBGand0.3 ±0.1,0.4 ±0.1,0.4 ±0.1,0.5 ±0.1,0.8 ±0.1 and 1.5 ±0.2 mm, respectively, after administration of tyramine; the corresponding values for the timerequired to reach baseline heart rate again were 1.4 ±0.6,1.2±0.4,9.2 ± 3.1,17.1± 3.5,>35 and >45 mm,respectively, after administration of MIBG and 1.0 ±0.1,1.0 ±0.2, 3.5 ±0.8, 6.9 ±1.7, 12.4 ±0.5 and 18.0 ±1.5mm, respectively, after administration of tyramine. This iswhy the preinjection heart rate before the last dose in thedose-responseexperiments shown in Figure 1A was higherthan the baseline heart rate in hearts exposed to MIBG(185 ±14 versus 156 ±10 bpm; P < 0.01) (Table 1)but didnot differ from baseline heart rate in hearts exposed totyramine (148 ±9 versus 156 ±9 bpm).

In other experiments, the responsesto 10 pmol MIBG andtyramine were followed for 60 mm (Fig. 3). The increase inheart rate lasted more than 60 mm after MIBG andapproximately20 mm after tyramineinjection(Fig. 3A).Accordingly, the norepinephrine overflow induced by MIBGand tyramine declined with half-lives of 57.8 ±13.4 and2.2 ±0.1 mm (P < 0.01), respectively (Fig. 3B). Fromtheresults shown in Figure 3B, the total norepinephrine overflow induced by 10 j.tmol MIBG (i.e., the area under thecurve from 0 mm to infinity) was calculated to be 79 ±19pmol/g, whereas the corresponding value for the totaloverflow evoked by tyramine was 259 ±44 pmol/g (P <

A0

FDoseof tyramineor MIBG (jm'iol)

FIGURE 1. Peak increasesin heartrateabovebaselineobservedin responseto seriesof consecutivelyadministeredMIBG ortyrammnedoses increasing from 0.03 to 10 pmol (A) and in response to single MIBG or tyramine dose of 10 pmol In absence (Co) orpresence(DE) of 2 pmoVLdesipramine(B). Isolated,spontaneouslybeating rabbit hearts perfusedwith Tyrode'ssolution(Langendoriftechnique,37°C,26 mL/min).MIBGand tyraminewere given as bolus injections(100 pL) into aortic cannula. Baselineheart rate was 164 ±4 bpm (n = 39). Shown are mean ±SEM of 7 (A), 8 (B; Co), 4 (B; DE, tyrammne)and 5 (B; DE, MIBG)observations.Fitting Hill's equationto meangroup resuftsgave these parameters(see Materialsand Methods):E,,,@59 (MIBG)and156 (tyrammne)bpm; ED@0.1 and 0.6 pmoVL;nH 0.5 and 0.9. Dose-responsecurves (A) were drawn to fit these parameters.*f@<0.01 for effect of desipramine.

SYMPAThOMIMETIC Ei@crs OF MIBG •Graefe et a!. 1345

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Tyramine

A B Tyramine MIBG250 J (10 pxnol)

200

150

100

50@

0.—--@ I I@CODE CoDE

D TyramineMIBG35. 1 (lOj.tmol)

30

25

20

15

10

5

A __@#@ .

CODE CODE

0It

0WO)z@C.-Q

Tyramine

FIGURE2. Totalincreasesinnorepinephrine(NE)(AandB)anddopamine(DA)(Cand 0) overflow observed in response toconsecutively administered MIBG or tyramine doses increasingfrom 0.1 to 10 pmol(AandC) andinresponsetosingleMIBGortyramine dose of 10 pmol in absence (Co)or presence (DE) of 2 pmoVLdesipramine(B and 0). Same experimentsas in Figure1. Baseline (i.e., spontaneous)NE and DAoverflow was 0.50 ±0.11 and 0.08 ±0.02pmoVg/min (n = 39 each), respectively.Shownare mean ±SEM of 7 (AandC), 8(B and D; Co), 4 (B and D; DE, tyramine)and 5 (B and D; DE, MIBG) observations.FittingHill's equationto meangroup resultsgave these parameters (see Materials andMethods):A, Em@35 (MIBG)and 218 (tyramine) pmol/g; ED@1.1 and I .8 pmoVL;nH1.0 and 1.1.; C, E@ 40 (tyramine)pmol/g;ED@5.7 pmovL;nH 1.2. Dose-responsecurves (A and C) were drawn to fit theseparameters.@ < 0.05 and **@< o@o@foreffect of desipramine on NE overflow.# =No detectable increase in DA overflow inpresenceof desipramine.

DoseoftyramineorMIBG(@imoI)

C35.

0

.@0.

DoseoftyramineorMIBG(iimol)

0.01). As far as the overflow response to tyramine isconcerned, this value was similar to those given in Figures2A and B. However, the overflow responses to 10 pmolMIBG shown in Figures 2A and B were smaller than thecalculated total overflow given above.

Cardlodepressant Effects of MIBGMIBG also acted as a cardiodepressant: A dose of 10 @.imol

markedly decreased heart rate and, to an even greater extent,LVPP(Fig. 4). These responsesto MIBGwere brief in onset,dosedependentand shortlastingand occurredat MIBGdoses O.3pmol(Table1).A 61%—88%decreasein LVPPwas also observed after injection of 10 @imolMIBG in twohearts paced at 5 Hz.

Injections of 100 pL deionized water or saline (used asvehicle solutions) did not alter heart rate or LVPP. However,the injection of 100 pL of 100 mmol/L sodium acetate (i.e.,the amount of acetate present in 10 pmol MIBG acetate)produced some decrease in heart rate and LVPP (Table 2).Nevertheless, Table 2 also shows that the cardiodepressant

effect of 10 pmol MIBG acetate was clearly more pronouncedthanthatof 10 @.unolsodiumacetate.Neithertheinjectionof deionizedwaterorsalinenortheinjectionof 10pmol sodium acetate caused any increases in norepinephrineor dopamineoverflow (datanot shown).

In hearts exposed to 2 pmol/L desipramine, the decreasein heart rate caused by 10 pmol MIBG was more pronouncedand lasted much longer than in hearts not exposed todesipramine (Table 2). The MIBG-induced decrease inLVPP, on the other hand, was attenuated in the presence ofdesipramine (Table 2). Desipramine, per se,had no effect onheart rate, but reduced LVPPby approximately 40% (Table 2).

Uptake by SK-N-SH CellsSK-N-SH cells were used to compare MIBG with tyra

mine and norepinephrine as substrates of uptake1. Thenisoxetine-sensitive uptake by these cells was taken toreflect membrane transport mediated by uptake1. The kineticanalysis of the saturation of uptake1 by these substratesshowed that MIBG, tyramine and norepinephrine (all in

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MIBGPreinjection,Time toTime toPreinjectionDecreaseTime toTimetodoseHADecreaseminimumpreinjectionLVPPinLVPPminimumpreinjection(pmol)(bpm)in

HA(%)HA (s)HA (5)(mm Hg)(%)LVPP (5)LVPP (s)

TABLE IDose-Dependent Cardiodepressant Effects of MIBG in Perfused, Isolated, Spontaneously Beating Rabbit Heart

0.3162±147±22.6±0.77±276±67±24.7±1.111±31166±156±13.5±0.911±476±715±35.7±0.714±13172±159±27.7±1.230±873±732±44.9±0.439±1410185

±14*19 ±4*18.6 ±35*115 ±13*69 ±764 ±3*5.8 ±0.795 ±28*

*P< 0.01forglobaldifferenceandlineartrendbetweenfourdosegroups,indicatingdose-dependenteffectof MIBGonbothHRandLVPP(analysisofvariancefor repeatedmeasures).

Givenaremean±SEMof sevenobservationseach.Bolusinjections(100 pL)containing0.3—10 pmolmetaiodobenzylguanidine(MIBG)were given consecutively (at intervals of 20—35mm) into aortic cannula of seven perfused rabbit hearts. Heart rate (HA)and leftventricularpulse pressure (LVPP)were monitoredbefore (preinjection)and after each MIBGdose.

DISCUSSION

This study deals with the sympathomimetic action ofMIBG in the isolated perfusedrabbitheart.Because it wasclear right from the beginning that MIBG would mainly actindirectly,theprototypeindirectlyactingsympathomimeticamine tyramine was included in this study. The resultsindicate that MIBG was indeed tyramine-like: The dosedependent increase in heart rate induced by MIBG wenthand in hand with a dose-dependent increase in norepinephrimeoverflow,withbotheffectsbeinghighlysusceptibletoinhibition by the uptake1 blocker desipramine.

Although tyramine-like, MIBG differed from tyramine intwo aspects. First, the duration of sympathomimetic action(at doses 1 Mmol) was longer for MIBG than for tyramineand, second, MIBG was much less effective in producingsympathomimetic effects than tyramine. The long durationof action of MIBG is readily explained by the fact thatMIBG, unlike tyramine, is not a substratefor monoamineoxidase. Therefore, MIBG is likely to disappear from theaxoplasm of noradrenergic neurons much more slowly thantyramine. The relatively low efficacy of MIBG could be aconsequence of the phenomenon of tachyphylaxis if thedegree of tachyphylaxis to MIBG's action were morepronounced than that to tyramine's action. In this experimental setting, however, tachyphylaxis to either MIBG ortyramine did not develop. This was substantiated by thefinding that the responses to the 10-pmol dose of either drugobserved at the end of the dose-response experiments did notdiffer from those observed after administration of thel0-j.imol dose to begin with.

The reason for MIBG being much less effective thantyramine may also reside in the complex mechanism bywhich indirectly acting sympathomimetic amines releasenorepinephrine from postganglionic sympathetic nerves. Allsuch agents are substrates for uptake1 and competitiveinhibitors of the vesicular monoamine transporter; theireffectiveness as norepinephrine releasers depends, above all,on the rate at which they are transported into the neuron aswell as the potency with which they block the vesicular

labeled form) had similar Km and Vm@values (Table 3). Theonly differencefoundin theseexperimentswasthatthe kvalue for the nisoxetine-resistant (nonsaturable) componentof uptakewas relativelyhigh for MIBG and tyraminecompared with norepinephrine (Table 3). The k value equalsthe slope of the regression line relating the 1-mm uptake inthe presence of nisoxetine (10 @imolIL)to the substrateconcentration and is probably related to the lipophilicity ofthe substrates.

Inhibition of Vesicular UptakeThe affinity of MIBG to the vesicular monoamine trans

porter was studied in membrane vesicles derived frombovine chromaffin granules. A 70%/30% mixture of epinephrine/norepinephrine (see Materials and Methods) was usedas substrate, and initial rates of uptake were measured atsubstrate concentrations ranging from 13 to 108 p.tmol/Lbothin the absenceandpresenceof variousconcentrationsofMIBG. As shown in Figure 5, MIBG reduced the vesicularuptake of 3H-catecholamines in a concentration-dependentmanner.Therewasa parallelshiftof theinhibitioncurvetothe right whenthe substrateconcentrationwas increased,thus suggesting that the inhibition of uptake produced byMIBG was competitive in nature.This was confirmed byfurther kinetic analysis of the results of Figure 5. The slopeof the double-reciprocal plot of the uptake rate (1/v) versusthe 3H-catecholamineconcentration(l/S) increasedwithincreasingMIBG concentration,whereastheordinateintercept of this plot remained unchanged. Moreover, the replotof theslopeof the1/vversus1/Splotagainsttheconcentration of MIBG revealed a straight line with a coefficient ofdetermination (r2) of 0.998. IC50 values for MIBG obtainedfrom the results shown in Figure 5 were transformed to Kvalues(n = 19);themeanK,was20(95%confidencelimits:17 and 23) pimol/L.

The K of tyramine for inhibition of the vesicular monoamine transporter determined previously under identicalexperimental conditions (23) was 2.5 (1.8 and 3.6) jimollL.Hence, tyramine is eight times more potent than MIBG ininhibiting the vesicular uptake of catecholamines.

SYMPATHOMIMETICEFFECTS OF MIBG •Graefe et al. 1347

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1!

0.1@

1

CEuI

200150100@

the inward transport of indirectly acting amines to theoutward transport of axoplasmic norepinephrine (25).

With these considerations in mind, we comparedMIBGand tyramine as substrates of uptake1 in SK-N-SH cells andasinhibitorsof the vesicularuptakeprocessin membranevesicles derived from bovine adrenomedullary chroma.ffingranules. SK-N-SH cells are known to express the uptake1transporter (26,27). The results obtained in these cells showthat the saturation of uptake1 by MIBG and tyramine ischaracterized by similar Km and V@ values, indicating that,at any given substrate concentration, rates of neuronaluptake are similar for the two compounds. Hence, ourfinding that MIBG was a poor norepinephrine releasercompared with tyramine cannot be explained by differencesbetween the rates of neuronal uptake of these two agents. Onthe other hand, as far as the inhibition of the vesicularmonoamine transporter is concerned, MIBG was eight timeslesspotentthantyramine.This observationexplainswhyMIBG was less effective than tyramine in releasing norepinephrine, because the ability of MIBG to mobilize storednorepinephrine by inhibiting the vesicular reuptake of thetransmitter is likely to be much less pronounced than that of

V. - - -

FIGURE 4. Representativetracingsshowtransientcardiodepressant effects of MIBG in isolated, perfused, spontaneouslybeating rabbit heart. Hearts were perfused with Tyrode's solution(37°C,26 mL/min),and left ventricularpressure(LVP), leftventricular pulse pressure (LVPP) and heart rate (HA) wererecordedfromsaline-filledlatexballooninsertedintoleftventricleand Isotecpressuretransducerconnectedto computer-assistedsignalprocessingsystem.Arrowindicatespointintimeat whichbolusof 10 pmol MIBG(100 pL)was injectedinto aorticcannula.

A

B

0

W .@ZE

a) 0.

I

MIBG

MIBG

0)IEETimeaftertyramineorMIBGinjection

(mm)

FIGURE 3. Time course of increasein heart rate (A) andnorepinephrine (NE) overflow (B) elicited by bolus injection of10 pmol MIBG or tyramine. Isolated, spontaneously beatingrabbit hearts perfusedwith Tyrode'ssolution (37°C,26 mL/min).Baseline heart rate was 165 ±7 bpm and spontaneous NEoverflow 0.36 ±0.07 pmoVg/min (n = 16 each). Shown arearithmetic(A)orgeometric(B)mean±SEMofeightobservations each. Regression lines (y = a + bx) shown in (B) weredrawnto fit theseequations:logy = 1.864 —0.141X(tyramine)and log y = —0.0793—0.00726X(MIBG).

reuptake of norepinephrine that normally leaks out of thetransmiuer storage vesicles at a rather high rate (24,25).Synaptic vesiclesfunction like a “pumpand leak system―(21) and store norepinephrine by generating a dynamicequilibrium between vesicular uptake (active inwardpump)anddiffusionout of the vesicles(passiveoutwardleak). Indirectly acting agents disarrange this dynamicequilibrium: They mobilize vesicularly stored norepinephrimeby competing with norepinephrine for vesicular reuptakeand induce a carrier-mediated transport of axoplasmicnorepinephrine out of the neuron. This type of substrateinduced norepinephrine release is part of a phenomenoncalled “facilitatedexchange diffusion,―in which the neuronal norepinephrine transporter responsible for uptake1 couples

80

6O@ LVPPC)

E20•

300

250

50@1@

117 118mm

119 120116

1348 THE Joui@uu.. OF NUCLEARMEDICINE •Vol. 40 •No. 8 •August 1999

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MIBGBaselineDecreaseTime toTime toBaselineDecreaseTime toTimetodoseDEHRinHRminimumbaselineLVPPinLVPPminimumbaseline(pmol)(pmovL)

n (bpm)(%)HR (s)HA (s)(mm Hg)(%)LVPP (s)LVPP (s)

KmVmax (pmol/mgk(pmol/mgprotein/min/Amine(pmol/L)protein/mm)pmoVL)

*P < 0.01 comparedwith 125l-metaiodobenzylguanidine(MIBG)and14C-tyramine(analysisofvariancefollowedbyBonferronitestformultiplecomparisons).

Given are mean ±SEM of three experiments carried out induplicate.The 1-mmcellularuptakeof labeledcompounds(atconcentrations ranging from 0.03 to 10 pmol/L)was determined inabsence (total uptake) and presence (nonmediated uptake) of 10pmol/Lnisoxetine(aselectiveuptake1blocker),andsaturableuptakemediatedby uptake1was calculatedfrom differencebetweentotaland nonmediated uptake.

TABLE 2Effect of Desipramine (DE) on Cardiodepressant Effect of 10 pmol MIBG in Perfused, Isolated,

Spontaneously Beating Rabbit Heart

006157±76±29.2±1.933±875±514±33.7±0.213±11009173±1111±1*12.3±2.858±1568±480±2t5.4±0.4152±43*1025168±1221±lf21.6±2.4@242±33t41±7@60±2@10.0±1.9@115±38

*P < 0.05andtP < 0.01whenheartsnotexposedto DEandinjectedwithMIBGwerecomparedwiththoseinjectedwithvehicle.:tP < 0.01and§P< 0.05whenheartsinjectedwithMIBGandexposedto DEwerecomparedwiththosenotexposedto DE(analysisof

variancefollowedbyBonferronitestfor multiplecomparisons).Givenare mean ±SEMofn observations. A100-pLbolus containing 10 pmolsodium acetate (0 = vehicle)or 10 pmolmetaiodobenzylgua

nidine(MIBG)acetatewas injectedintoaorticcannulaof perfusedrabbithearts.Heartrate(HA)and leftventricularpulsepressure(LVPP)were monitored before (baseline) and after vehicle or MIBGadministration. In some experiments, 2 pmol/LDE was present in mediumperfusing the hearts.

tyramine. In this context it must be emphasized that there aretwo isoforms of the vesicular monoamine transporter(VMAT1 and VMAT2) and that the main isoform operativein bovine chromaffin granules (VMAT2) is also expressed insynaptic vesicles of postganglionic sympathetic neurons(28,29).

The doses of MIBG used in this study cannot easily beextrapolated to give MIBG concentrations in the mediumperfusing the hearts. Nevertheless, sympathomimetic effectsof MIBG such as increases in heart rate and blood pressurehave not been reported as common findings in the clinicalsetting. In other words, the plasma concentrations observedafter intravenous administration of therapeutic doses of‘311-MIBG(MIBG mass 25—35pmol; plasma concentration:s0.l pmol/L [2,30]) must be less than sympathomimeticallyeffective MIBG concentrations. Even MIBG doses of 255—

TABLE 3Saturation Constants (K,@and Vm@x)for Uptake1 and RateConstant(k) for Nonmediated(Nonsaturable)Uptakeof

LabeledMIBG, Tyramineand Norepinephrineby SK-N-SHCells

290 pmol, which produced plasma concentrations of approximately 1 @imol/Lwhen given by intravenous infusion within3 h, were not reported to be associated with clear sympathomimetic effects (31). This is in agreement with preliminary

FIGURE 5. Inhibitionby MIBG of vesicular3H-catecholamineuptake. Membrane vesicles obtained from bovine chromaffingranules were exposed to 12.7 ±0.8 (•),30.9±0.7 (•)and108.4 ±6.9 (S) pmoVL of 3H-catecholaminemixture (seeMaterials and Methods) and initial rates of reserpine-sensitive3H-catecholamineuptake were measured both in absence andpresence of MIBG (3—1000pmol/L). Control rates of uptakeobservedat substrateconcentrationsof 13, 31 and 108 pmol/Lwere3.7 ±0.5 (n = 6), 5.5 ±0.3 (n = 7) and6.6 ±0.3 (n = 6)nmoVmgprotein/ mm, respectively.Shown are mean ±SEM ofsix(S), seven(•)andsix(•)observations.WhenHill'sequationwas fitted to mean group data, the following parameters wereobtained(seeMaterialsandMethods):lC@(fromlefttoright)=41 .6, 67.7 and 166.4 pmoVL; l@ = 98.3%, 95.3% and 92.9%;nH = —1.02, —1.04 and —1.15. Concentration-effect curvesshowningraphweredrawntofittheseparameters.

a)

0.

CE@

0@.C @-C-)a)— I.C@0

C!)

C@

C-)C,)a)>

Concentrationof MIBG(pmol/L)

1@l-MlBG1.6 ±0.843.4 ±10.110.2 ±1.114C-tyramineI.7 ±0.536.6 ±2.99.9 ±0.73H-norepinephnne1.0 ±0.235.6 ±3.73.2 ±0.7*

SYMPAThOMIMETICEwi@crs OF MIBG •Graefe et a!. 1349

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findings that, in the perfused rabbit heart, 1 pmol/L M@IBGwas a threshold concentration for the positive chronotropiceffectof thedrug(Bossle,WölfelandGraefe;unpublishedobservations).

Female,athymic,nudemiceinjectedintravenouslywith10 @.imo1MIBG died immediately after the injection (32).This finding might have been due to the indirect sympathomimetic effects of MIBG. Alternatively, the mice may havedied as a resultof the cardiodepressantactionof MIBGobserved in this study. This action may be related to MIBGacting as a reversible inhibitor of the mitochondrial respiration (33,34). The cardiodepressant effects manifested themselves in initial short-lived decreases in heart rate and LVPP.Onlywhentheheartrateisheldconstant,thevalueof LVPPreflects the inotropic state of the left ventricle. Because themarked decrease in LVPP was observed not only in spontaneously beating but in paced hearts, it can be concluded thatMIBG compromises both the pacemakerautomaticityandthe myocardialcontractility.The transientnatureof thecardiodepressant effects of MIBG was obviously a consequenceof thewayMIBG wasadministered.Bolusinjectionsinto the aorticcannulado not producesustainedMIBGconcentrations in the perfusion medium and, hence, will notbring about sustained cardiodepressant effects.

BecauseMIBG's cardiodepressantandsympathomimeticactions were observed in the same dose range, it can beinferred that the negative chronotropic and inotropic effectsof the drug are unlikely to become apparent in the clinicalsetting, just as the occurrence of the sympathomimeticeffectsof MIBG is uncommonin patientsgivendiagnosticor therapeuticdosesof radioiodinatedMIBG (seeabove).One may even envisage the possibility that the sympathomimeticeffectsand initial cardiodepressantof MIBG maskeachother.As far as the negativechronotropicactionofMIBG is concerned, this possibility was verified in thisstudy by the finding that desipramine, which inhibited thedrug-induced norepinephrine release, enhanced the initialheart rate decrease caused by MIBG and greatly prolongedthedurationof thisdrugaction.

CONCLUSION

In spontaneously beating, isolated, perfused rabbit hearts,MIBG was tyramine-like in that it behaved as an indirectlyacting sympathomimetic agent. It was much less effectivethan tyramine in releasing endogenous norepinephrine,becauseits ability to mobilize norepinephrinefrom thetransmitter storage vesicles was much less pronounced thanthat of tyramine. The duration of action of high doses ofMIBG was much longer than that of tyramine. This is aconsequenceof the fact that MIBG is clearedfrom theaxoplasm of noradrenergic neurons much more slowly thantyramine, because, unlike tyramine, MIBG is not metabolizedbymonoamineoxidaseandbecauseMIBG istakenupby synaptic vesicles much less avidly than tyramine. Incontrast to tyramine, MIBG also had cardiodepressanteffectsthat were observedbeforethe occurrenceof the

drug's sympathomimetic effects. Neither the sympathomimetic nor the cardiodepressant effects of MIBG are likely tocome into play in patients given diagnostic or therapeuticdoses of radioiodinated MIBG not exceeding 4 pmol/kg.

ACKNOWLEDGMENTS

The authors thank Marianne Babl and Angelika Keller fortechnical assistance. This work was supported by theDeutsche Forschungsgemeinschaft (Gr. 490/10).

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SYMPATHOMIMETIC Ewi@crs OF MIBG •Graefe et a!. 1351

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1999;40:1342-1351.J Nucl Med.   Farahati and Heinz BönischKarl-Heinz Graefe, Franz Bossle, Reinhard Wölfel, Albrecht Burger, Maria Souladaki, Dirk Bier, Klaus Dutschka, Jamshid  Sympathomimetic Effects of MIBG: Comparison with Tyramine

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