everal new technetium-99m (@mTc) agents for re gional myocardial perfusion imaging have been re ported (1-8). These agents are potential substitutes for thaffium-201 (2Ofrfl)as the major radiopharmaceutical for routine nuclear medicine application. The @â€oeTc labeled isonitriles, initially developed by Jones and Davison, have reached the final stage of clinical trial (1). The first agent of this series was [@â€oeTc]TBI (t butylisonitrile), which showed very high myocardial uptake and retention reflecting regional perfusion. However, the initial lung uptake and the subsequent high liver retention of [99mTcJTBIclearly indicates the need for further researchand development (2). A new generation of isonitriles, [@mTc]MIBI (2-methoxyiso butylnitrile) and CPI (2-carboxypropanylisonitrile) which show improved liver and lung washout, and the same high myocardial uptake and prolonged retention was reported (3-5). The [99mTcIMIBIiscurrently under phase II clinical trial. New boronic acid adducts of vicinyl dioximes, â€oecage― complexes of @Tc, (BATO, Squibb, SQ-302l7) have been reported (6). Initial cm ical study has also indicated that they may be useful as myocardial perfusion tracers (7). Another group of Tc. (Arene)2+ compoundS, which is a â€oesandwich― complex, wasreported(8). The clinicalevaluationofthese agents Received May 18, 1988;revision accepted Oct. 13, 1988. For reprintscontact: Hank F. Kung, PhD, Div. of Nuclear Medicine, Dept. of Radiology, University of Pennsylvania, 3400 Spruce St., Philadelphia, PA 19104. in humans has not yet been reported; nevertheless, initial studies in animals showed very high myocardial uptake and prolonged retention. Recent advances in chemistry of @Tccomplexes based on N2S2 ligands has dramatically enhanced our ability to predict the chemical structure of the final 99mTc complexes. This series of ligands forms strong complexes with (Tc = @ The x-ray crystallography studies of several N2S2 complexes developed by our selves and others (9-19) has confirmed the (Tc = O)@@ chemical state and the pyramidal core structure. In dium-l 11(â€oeIn) is a radionucide with a T,a 2.8 days and gamma rays of 172 and 247 keV, which are suitable for nuclear medicine imaging studies. Currently, the radionucide [â€oeIn]oxine is being used for white blood cell (20) and monoclonal antibody labeling. In order to investigate further the radiochemistry of indium, we have initiated a study using the N2S2ligand, tetraethyl bis-(aminoethanethiol) (TE-BAT), for complexing In@3. This paper presents our data on synthesis, radiolabeling, characterization, and biodistribution of this unique In complex. For convenience in this study, ‘ I3mIneluted from a tin-l 13m indium-l l3m (IL3mSn@I@3mIn) genera tor was employed as the tracer. However, for imaging studies, ‘ ‘ ‘In is more suitable. MATERIALS AND METhODS General The preparation of TE-BAT was achieved by a method reported previously (10). The only difference is that lithium 367 Volume30 • Number3 • March1989 Synthesis, Characterization, and Biodistribution of [ll3mln]TEBAT. A New Myocardial Imaging Agent B.L. Liu, H.F. Kung, Y.T. Jin, L. Zhu, and M. Meng Department ofChemistry, Beijing Normal University, Beijing, China; and Department of Radiology, University ofPennsylvania, Philadelphia Inorderto developa newmyocardialperfusionagent,newlipid-solublecomplexescontaining a netchargeof+1 wereevaluated. Synthesis, radiolabeling, characterization, and biodistnbution of a unique indium complex, [11@―lnJTE-BAT (tetraethyl-bis-aminoethanethiol), are descilbed.The complexformationbetweenln'@3 and TE-BAT ligandis rapid,simple,and of highyield(@95%). Thisprocessis amenableto kit formulation.The complexhasa net chargeof+1 andanIn/ligand ratioofI :1. Biodistilbution inmiceshowshigherheartuptake and longerretentionas comparedto @°@TI. This complex,when labeIed@with 111ln, shows promiseasa possibletracerformyocardialperfusionimaging. J Nucl Med 30'367—373, 1989 by on April 4, 2019. For personal use only. jnm.snmjournals.org Downloaded from
Transcript
everal new technetium-99m (@mTc) agents for regional myocardial perfusion imaging have been reported (1-8). These agents are potential substitutes forthaffium-201 (2Ofrfl)as the major radiopharmaceuticalfor routine nuclear medicine application. The @“Tclabeled isonitriles, initially developed by Jones andDavison, have reached the final stage of clinical trial(1). The first agent of this series was [@“Tc]TBI(tbutylisonitrile), which showed very high myocardialuptake and retention reflecting regional perfusion.However, the initial lung uptake and the subsequenthigh liver retention of [99mTcJTBIclearly indicates theneed for further researchand development(2). A newgeneration of isonitriles, [@mTc]MIBI (2-methoxyisobutylnitrile) and CPI (2-carboxypropanylisonitrile)which show improved liver and lung washout, and thesame high myocardial uptake and prolonged retentionwas reported (3-5). The [99mTcIMIBIis currently underphase II clinical trial. New boronic acid adducts ofvicinyl dioximes, “cage―complexes of @Tc,(BATO,Squibb, SQ-302l7) have been reported (6). Initial cmical study has also indicated that they may be useful asmyocardial perfusion tracers (7). Another group of Tc.(Arene)2+ compoundS, which is a “sandwich―complex,wasreported(8). The clinicalevaluationofthese agents
ReceivedMay 18, 1988;revision accepted Oct. 13, 1988.For reprintscontact:Hank F. Kung, PhD, Div. of Nuclear
Medicine, Dept. of Radiology, University of Pennsylvania, 3400Spruce St., Philadelphia,PA 19104.
in humans has not yet been reported; nevertheless,initial studies in animals showed very high myocardialuptake and prolonged retention.
Recent advances in chemistry of @Tccomplexesbased on N2S2 ligands has dramatically enhanced ourability to predict the chemical structure of the final99mTccomplexes. This series of ligands forms strongcomplexes with (Tc =@ The x-ray crystallographystudies of several N2S2complexes developed by ourselves and others (9-19) has confirmed the (Tc = O)@@chemical state and the pyramidal core structure. Indium-l 11(“In)is a radionucide with a T,a 2.8 daysand gamma rays of 172 and 247 keV, which are suitablefor nuclear medicine imaging studies. Currently, theradionucide [“In]oxine is being used for white bloodcell (20) and monoclonal antibody labeling. In order toinvestigate further the radiochemistry of indium, wehave initiated a study using the N2S2ligand, tetraethylbis-(aminoethanethiol) (TE-BAT), for complexing [email protected] paper presents our data on synthesis, radiolabeling,characterization, and biodistribution of this unique Incomplex. For convenience in this study, ‘I3mInelutedfrom a tin-l 13m indium-l l3m (IL3mSn@I@3mIn)generator was employed as the tracer. However, for imagingstudies, ‘‘‘Inis more suitable.
MATERIALS AND METhODSGeneral
The preparation of TE-BAT was achieved by a methodreported previously (10). The only difference is that lithium
367Volume30 •Number3 •March1989
Synthesis, Characterization, and Biodistributionof [ll3mln]TEBAT. A New MyocardialImaging AgentB.L. Liu, H.F. Kung, Y.T. Jin, L. Zhu, and M. Meng
Department ofChemistry, Beijing Normal University,Beijing, China; and Department ofRadiology, University ofPennsylvania, Philadelphia
In orderto developa new myocardialperfusionagent, new lipid-solublecomplexescontaininga netchargeof+1 wereevaluated.Synthesis,radiolabeling,characterization,andbiodistnbution of a unique indium complex, [11@―lnJTE-BAT(tetraethyl-bis-aminoethanethiol),are descilbed.The complexformationbetweenln'@3andTE-BAT ligandis rapid,simple,andof highyield(@95%). Thisprocessis amenableto kit formulation.The complexhas a netchargeof+1 andanIn/ligandratioof I :1. Biodistilbutioninmiceshowshigherheartuptakeand longerretentionas comparedto @°@TI.Thiscomplex,when labeIed@with111ln,showspromiseas a possibletracerfor myocardialperfusionimaging.
J Nucl Med 30'367—373,1989
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aluminum hydride was employedfor the last reduction stepof diimine intermediate(19). The dimercapto hydrochloridesalt of TE-BAT was precipitated and used for this study.Indium-l 13mwasobtainedbyelutinga' ‘3Sn-'l3mIngenerator(Institute ofAtomic Energy, Beijing, China) with 0.1 N Ha.
RadiolabelingNo-carrier-added[‘sImm]chloride(1 mCi/mi) eluted with
0.1 N H@ was added to a test tube containing the BAT-TEligand (2 mg) in 1 ml of water. In order to maintain the pHat 4-5, a simultaneousaddition ofa solutionof 5%NaOH inthe reactionmixtureis needed.The mixturewasvortexedandkept in a water bath at 80C for 0.5 hr. The percent labelingyield was measured by thin layer chromatography (TIC)(Silicagel plate, developingsolvent:acetone, R@= 0.6). Theradiochemical purity usually was over 96%. This material wasused directly for animal studies. The effect of acidity andreaction time on the formation of this complex was determined by the same TLC technique.
Characterization of III-TE-BAT complexDetermination of composition. The composition of the
complex, [lI3mInJTh..BAT,was determinedby a pH titrationmethod (Radiometer, PH64). The formation of this complexfollows the equation:
TE-BAT + @@3mIn+3.@[Il3mInThBAT]++ 2W.
When [‘l3mIfl]TEBAT is formed, two equivalents of [W]are released and can be titrated by a standardized sodiumhydroxide solution (0.OlN)(Fig. 1).The titration is performedunder two different conditions (with or without [In@3]):solution (A) containing 1.0mg (0.391 mM) of TE-BATin 7 mlof 1 mM HQ solution and solution (B) containing the sameamount ofligand, 1.0mg ofTE-BAT in 3 ml of 1 mMHQ,and 4 ml ofln(NO3)3solution(1.9 mg in 50 ml of 1mM HQ,1.26 x i0-@mM). Both ofthe solutions contain 0.1 N of NaCI(the same ionic strength). Based on the difference betweentitration curves A and B, the formation function can becalculated (21)
8 —@‘ l3mIflTEBAT][TM]
where [TM] total concentrationof [email protected] the endpoint oftitration the formation function fi approaches unity, ifthe In!ligand ratio is equal to one.
Determination ofnet charge. Determination of net chargeof this complex was achievedby the ion exchangemethod
(21). Ion exchange resin (cation, 10 mg/each experiment) was
placed in a test tube together with a solution of [‘l3mIn]TE@BAT (5 ml, at pH 0.9-2.3). The mixture was shaken for 1 hr.The resinand the solutionwereseparated.The residualradioactivity in the solution was measured and the distributioncoefficient (D) was calculated by counts in resin/counts insolution.
[‘1ImInTEBATI+x+@@ R-[' I3mIn@TE@BAT]@@
RH: Cation exchange resinThe equilibriumconstant = K
K —@— [Il3mIflmBA'fl+x[@J4]
Distribution Coefficient = D
D —R-[―3mIn-TE-BA'fl'— [II3mIfl'@BAT]@
log D = log K + log[RH,j + xph log D = xph + C.
The relationshipbetweenlog D and pH is a straight lineand the slope,x, is equal to the net chargeofthe complex.
BiOdistribUtiOn In Mice
Biodistributionof [hl3mIn]TE@BATwas studied in malemice (18-22 g) which were allowed access to food and waterad lib. Saline solution containing[' IlmIn]TE@BATin a volumeof 0.1 ml was injected directly into the tail vein. Mice werekilled(at varioustime points,2 mm to 1hr, postinjection)bycardiac excision under ether anesthesia. The organs of interestwere removed and counted using a well gamma counter.Percent dose per organ was CalCUlatedby comparison of tissuecounts to suitably diluted aliquots of injected material. Totalactivities of blood and muscle were CalCUlatedassuming thatthey are 7% and 40% oftotal bodyweight,respectively.
RESULTS
Characterization of I―MnITE-BATEffects of acidity. It is well known that In(OH)3 is
the predominant form when the pH of the reaction ishigher than 5. The formation of the complex was evaluated at various pHs to determine the optimum conditions for labeling. The results shown in Figure 2suggest that the labeling yield reaches a plateau betweenpH 4 and 5. At higher pH, precipitation of the ligand,
+1
113m ,@
HN HN\ I
FiGURE 1Chemical equation for the formation of [11@in]TE-BAT.
368 Liu,Kung,Jinetal TheJournalof NuclearMedicine
Ig@
%InS
.5 p14,
NH
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owing to its limited solubility in water, is observed (Fig.2).
Reaction time. At pH 4, the labeling yield was evaluated at various reaction times. The formation of thecomplex reachesa plateau at 20 mm (Fig. 3). Prolongedheating appears to have no significant effect on thelabeling yield.
Determination ofcomposition ofln-TE-BAT. As indicated in Figure 1, the formation of no-carrier-added“3mIn-TE-BATproduces two hydrogen ions. After thecomplexation, the pH ofthe reactionsolution decreases.The decrease of pH is stoichiometrically proportional
to the formation of the complex. This change can bemeasured by using acid-base titration techniques. Thetitration curves for the TE-BAT ligand at the sameconcentration with (B) and without (A) the presence ofindium metal ion are represented in Figure 4. Fromthis figure the concentration of[H―]can be calculated.The ionic strengthofthe solutions, under which curvesA and B are generated, is the same, except that solutionB contained In'3 (1.00 x lO@).At the same pH value,curves A and B show that a different volume of sodiumhydroxide is consumed. The difference is a reflectionofcomplex formation, and can be employed to calculate
bt
V1)
a)U
Ia)
Ua)Va)
90
80
70
60
FIGURE 3Effectsof time on the formationof[11@'in]TE-BAT.Theformationof thecomplex reaches a plateau at 20 mm.
5010 20
Time (mm)
0
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FiGURE4Thetitrationcurvesof the ligand:TEBAT, with (B) and without(A) thepresenceof indiummetalion(1.00 x10-@ M). The difference betweenthese two curves at the same pHvalue is stoichiometricallyproportionalto the complexformation.
32.9 3.4 3.5 3.6
Vol (mL)
the concentration ofthe complex. Basedon the titrationcurves and the stoichiometric relationship of hydrogenion release and complex formation, the formation function (n) can be calculated. The relationship of formationfunction and pH is representedin Figure 5. This figureclearly indicates that the composition ofthe complex is1:1, confirming the structure shown in Figure 1.
Determination of the net charge of the complex.
Using the ion exchange method, to determine the distribution constant (D) between resin and aqueous solution at various pHs, the net charge of the complexcan be determined based on the following equation:
log D = xpH@+C.
From Figure 6 the net charge, x, is determined to be1.17.It is most likely that the net chargeofthis complex
1.00.90.80.70.6.0.5.0.4
0.3 —3.0 3.5 4.0 4.5 5.0
FIGURE5The relationshipof the formationfunction (ñ)and pH. This figure mdicates that the compositionof thecomplex is 1:1, comfirming the themical structure shown in Figure 1.
I I I I
pH
370 Liu,Kung,Jinetal TheJournalof NuclearMedicine
A B
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is +1. This is one more piece of evidence suggestingthat the chemical structure in Figure 1 is correct.
Biodistribution in MiceAfter an i.v. injection of [hl3mInJTE@BATin mice, a
significant heart uptake (32.9% dose/g) at 2 mm (i.v.)was observed. The heart uptake dropped to 22.5% dose!gat 15 minand lO.l%dose/gat 1 hr(Table I and Fig.
Ea)I.
a)
a)
7). The uptakeat thesetime points is higher than thosereported for thaffium-201 (@°‘TI)and technetium-99m(@Tc) ThI (1, 21). The heart to lung, and heart toblood ratios for this complex are comparable or superiorto those reported for 20'TI and [@mTc]TBI.There issignificant uptake in liver andlung which washes outwith time. The ratios of heart/blood, heart/lung andheart/liver are reported in Table 1.
. Heart0 Lung
U Liver
0 Blood
50
40
30
20
10
0
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The complex formation between In and TE-BATligand is very rapid, simple and occurs in high yield(@95%).Thehighlabelingefficiencyandexcellentpurity of this labeling reaction means that it requiresno further purification before being used in animalstudies. It is possible that this process is amenable forkit formulation.
The labeling reaction is pH sensitive; the optimumpH range is between 4-5. This pH can be easily maintamed by the addition of buffer solution and is, therefore, easily adaptable for a simple one step reaction.The net chargeofthe no-carrier-added[hI3mIn]TE@BATis determined to be +1. In view of the fact that almostall ofthe myocardial perfusion imaging agents reportedare +1 charge molecules, it is not surprising that [‘I3mIn]TE-BAT, with the same net charge, also displays goodheart uptake and retention. In mice, this agent displaysfast myocardial uptake and rapid blood and lung washout; at 20 mm postinjection the heart/blood and heart!lung ratios reach 21 and 2.25, respectively. At 1 hrpostinjection the heart uptake still remains high:10. 13% dose/g. The heart uptake is comparable to thatreported for 20―fland [99mTc]TBI(23). The biologicbehavior of [1I3mInITEBATclearly suggests that thisagent is potentially useful for myocardial perfusionimaging. It is necessary to determine the chemical structure by preparing “cold―In-TE-BAT complexes. Inaddition, further studies in primates and humans areneeded, especially the “redistribution―of this agent inmyocardial tissue, to fully characterize the physiologicalproperties.
Technetium-99m-labeled myocardial perfusionagents are currently being developed, which could potentially replace the 201'fl,the agent being used in theclinics at present. However, despite the superior physical characteristics ofthe @mTcisotope (gamma ray 140
keV, T,,2 = 6 hr), the biologic behavior (no redistribution) of @Tcisonitriles is different from @°‘Tl(withdelayed redistribution). The lack of redistribution forthe 99mTcagents is being perceived as being less usefulthan 201T1,because the viability of damaged myocardium could not be studied effectively (24). This isprobably one of the most controversial issues for myocardial perfusion imaging. The In-complex reported inthis paper may have a different uptake and retentionpauern when labeled with “In(T,,@= 2.8 days); it mayoffer an alternative agent for evaluation of “redistribution―at 24 hr or even 48 hr after the initial injection.
In conclusion, ‘l3mln(III) chelates directly with TEBAT to give a +1 charged complex. Biodistribution inmice showed significant heart uptake, a long retentiontime, high heart to blood ratios and low liver uptake.This agent, when labeled with ‘‘‘In,shows promise asa possible radiotracerfor myocardialperfusion imaging.
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