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Underscoring the Inuence of Inorganic Chemistry on NuclearImaging with RadiometalsBrian M. Zeglis, Jacob L. Houghton, Michael J. Evans, Nerissa Viola-Villegas, and Jason S. Lewis*

Department of Radiology and the Program in Molecular Pharmacology and Chemistry, Memorial Sloan-Kettering Cancer Center,New York, New York, United States

ABSTRACT: Over the past several decades, radionuclides have matured from largelyesoteric and experimental technologies to indispensible components of medicaldiagnostics. Driving this transition, in part, have been mutually necessary advances in

biomedical engineering, nuclear medicine, and cancer biology. Somewhat unsung hasbeen the seminal role of inorganic chemistry in fostering the development of newradiotracers. In this regard, the purpose of this Forum Article is to more visibly highlightthe signicant contributions of inorganic chemistry to nuclear imaging by detailing the development ofve metal-based imagingagents: 64Cu-ATSM, 68Ga-DOTATOC, 89Zr-transferrin, 99mTc-sestamibi, and 99mTc-colloids. In a concluding section, severalunmet needs both in and out of the laboratory will be discussed to stimulate conversation between inorganic chemists and theimaging community.

INTRODUCTION

Over the past 3 decades, nuclear imaging modalities haverevolutionized clinical medicine, particularly cardiology, neu-rology, and oncology.1,2 Indeed, the ability of positron emissiontomography (PET) and single photon emission computedtomography (SPECT) to provide functional and biochemicalinformation about tissues to complement the anatomical mapsprovided by other imaging modalities has proven vital in thediagnosis and management of disease. The advent of molecular

imaging has in large part been due to remarkable advances inbiomedical engineering, medical physics, halogen radiochemistry,and cancer biology. Yet the critical role of inorganic chemistry inthe rise of nuclear imaging has often become lost in the margins.In the following pages, we will seek to remedy this oversight. We

will rst discuss the intersection of inorganic chemistry,radiochemistry, and nuclear imaging in general terms. Then, atgreater length, we will use ve particularly effective or promisingmetal-based imaging agents as case studies both to illustrate thefundamental role of inorganic chemistry in the development ofradiopharmaceuticals and to more visibly celebrate thecontributions of inorganic chemistry to nuclear imaging.

Why Use a Metallic Radioisotope? Before we delve anydeeper into our discussion, we must rst answer one simple

question:

Why use a metallic radioisotope?

This question be-comes especially important when considering that PET imagingis largely dominated by a radiohalogen, uorine-18 (18F,t1/2 109.8 min). The answer is straightforward: radiometals provideexibility, modularity, and facility unmatched by other imagingisotopes.

First, the wide variety of metallic radionuclides allows for theprecise tailoring of the physical half-life of the radioisotope tothe biological half-life of the targeting vector (Figure 1). Forexample, agents with short in vivo residence times can belabeled with gallium-68 (68Ga; t1/2 68 min) or technetium-99m (99mTc; t1/2 6 h), while vectors that require longeramounts of time to reach their target can be labeled with

copper-64 (64Cu;t1/2 12.7 h), yttrium-86 (86Y;t1/2 14.7 h),

indium-111 (111In; t1/2 2.8 days), or zirconium-89 (89Zr;

t1/2 3.2 days) (Figure1 and Tables1 and 2).37

Second, the simplicity and modularity of using differentbifunctional chelators and radiometals facilitate the creation of awide variety of imaging agents. For example, with relative ease,the same antibody can be conjugated to the chelatorsdesferrioxamine (DFO), diethylenetriaminepentaacetic acid(DTPA), and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic

acid (DOTA) for labeling with

89

Zr for PET imaging,

111

In forSPECT imaging, or lutetium-177 (177Lu) for radioimmunotherapy.In some cases, particularly with the versatile chelators DOTA,DTPA, and 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA),the radiometal may be exchanged without changing the chelator atall. Either way, this modularity becomes especially clinically useful

when an imaging agent labeled with one isotope can be used asacompanion diagnostic tool for a therapeutic agent bearing another.8

Third, generally speaking, radiometalation reactions are rapidand can be achieved under mild conditions. Puricationprocedures are also quite simple, typically involving cation-exchange chromatography or reverse-phase C18cartridges. It isin this area that radiometals likely offer the greatest advantageover radiohalogens because probes bearing the latter often

require multistep syntheses, harsh reaction conditions, andcomplicated purications.Fourth, many radiometalsfor example, 86Y, 89Zr, and

111Inare known to residualize inside cells following theuptake of their vector, resulting in increased retention of theradioactivity inside the target tissue and higher tumor-to-

background activity ratios than nonresidualizing radiohalogenssuch as 18F, iodine-124 (124I), and bromine-76 (76Br).9

Special Issue: Imaging and Sensing

Received: June 25, 2013

Forum Article

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Finally, yet no less critically, metallic radioisotopes present atremendous opportunity to expand the availability of imagingagents beyond hospitals with nearby cyclotron facilities because

many radiometals can be produced via portable generatorsystems (e.g., 68Ga and 99mTc) or possess physical half-liveslong enough such that they can be shipped to research

Figure 1.Illustration of the variety of metals with isotopes suitable for nuclear imaging. Elements with isotopes suitable for PET are color-codedblue, and elements with isotopes suitable for SPECT are color-coded red. The shading corresponds to half-life, with longer half-lives darker andshorter half-lives lighter. Elements with multiple shadings have multiple isotopes suitable for imaging.

Table 1. Physical Properties of Some Common PET Radiometalsa

isotope half-life/h sourceproduction

reactiondecay mode

(% branching ratio) E+/keVabundance,

I+/%E/keV

(intensity, I/%)relevant

oxidation states

commoncoordination

numbers64Cu 12.7 cyclotron 64Ni(p,n)64Cu + + (61.5) 278.2(9) 17.60(22) 511.0 (35.2) 1+, 2+ 4, 5, 6

+ (17.6)

(38.5)68Ga 1.1 generator 68Ge/68Ga + + (100) 836.02(56) 87.94(12) 511.0 (178.3) 3+ 4, 5, 6

+ (89.1)86Y 14.7 cyclotron 86Sr(p,n)86Y + + (100) 535(7) 11.9(5) 443.1 (16.9) 3+ 8, 9

+ (31.9) 511.0 (64)

627.7 (36.2)

703.3 (15)

777.4 (22.4)

1076.6 (82.5)1153.0 (30.5)

1854.4 (17.2)

1920.7 (20.8)89Zr 78.4 cyclotron 89Y(p,n)89Zr + + (100) 395.5(11) 22.74(24) 511.0 (45.5) 4+ 8

+ (22.7) 909.2 (99.0)aUnless otherwise stated, standard deviations are given in parentheses (IT = isomeric transition; = electron capture).208

Table 2. Physical Properties of Some Common SPECT Radiometalsa

isotope half-life/h sourceproduction

reactiondecay mode (% branching

ratio) E/keVabundance,

I/%relevant oxidation

statescommon coordination

numbers67Ga 78.2 cyclotron natZn(p,x)67Ga (100) 91.265(5) 3.11(4) 3+ 4, 5, 6

68Zn(p,2n)67Ga 93.310(5) 38.81(3)

184.576(10) 21.410(10)208.950(10) 2.460(10)

300.217(10) 16.64(12)

393.527(10) 4.56(24)99mTc 6.0 generator 99Mo/99mTc (0.0037) 140.511(1) 89.06 1to 7+ 4, 5, 6

IT (99.9963)111In 67.3 cyclotron 111Cd(p,n)111In (100) 171.28(3) 90.7(9) 3+ 5, 6, 7, 8

245.35(4) 94.1(10)aUnless otherwise stated, standard deviations are given in parentheses (IT = isomeric transition; = electron capture).208

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laboratories and hospitals without excessive decay (e.g., 64Cu,111In, and 89Zr).

Production and Purication of Radiometals. The rststep in the synthesis of a radiometal-based imaging agent isproduction of the radiometal itself. Radiometals can beproduced via three distinct routes: decay of longer-livedradionuclides in a generator, nuclear bombardment reactions

in a cyclotron, or nuclear bombardment reactions in a nuclearreactor (see Tables1and 2). 68Ga, for example, is formed viaelectron capture decay of its parent radionuclide, germanium-68 (68Ge), and thus can be produced using a compact, cost-effective, and convenient 68Ge/68Ga generator system. 64Cu,in contrast, can be produced either on a nuclear reactor[via the 63Cu(n,)64Cu or 64Zn(n,p)64Cu reaction] or, farmore commonly, by use of a biomedical cyclotron via the64Ni(p,n)64Cu reaction.10 As an aside, it is important to notethat each of these isotopes emits radiation other than the posi-trons and photons useful for imaging. Some of these emissions,such as the variety of high-energy photons from 86Y and the909 keV photon from 89Zr, require special consideration withregard to handling, shielding, and dosimetry.11

Yet the process does not end with the creation of the desiredradiometal. The radiometal must be puried from its parentisotope and other byproducts of the nuclear reaction andisolated in a useful form prior to its incorporation into animaging agent. Here lies the rst point of intersection betweeninorganic chemistry and radiochemistry.

86Y, for example, is most often produced via the 86Sr(p,n)86Yreaction by the proton bombardment of [86Sr]-enriched SrCO3or SrO targets on a cyclotron. A variety of different techniqueshave been employed to separate the 86Y3+ cation from thetarget and byproducts, including cation-exchange chromatog-raphy, cation-exchange chromatography followed by coprecipi-tationwithLaIII or FeIII, and chromatography using Sr-selectiveresins.12,13 Recently, a particularly effective and economicalmethod for isolating 86Y using electr olysis has beendeveloped.14 After irradiation of a [86Sr]-enriched SrO targetcoated onto a platinum disk, the entire target is dissolved innitric acid with NH4NO3as an electrolyte. This solution is thenplaced in an electrochemical cell in which two successiverounds of electrolysis are employed to separate 86Y fromresidual Sr via electrodeposition on a platinum-wire electrode.This 86Y-coated platinum wire electrode can then be removedfrom the cell and washed with EtOH and HNO3. This solutioncan then be evaporated and reconstituted in 0.1 M HCl to yield86Y3+ in very high specic activity and radionuclidic purity.Importantly, this method also allows for the efficient recyclingof the expensive, isotopically enriched 86Sr target material.

In another example, 89

Zr is produced via the 89

Y(p,n)89

Zrreaction by proton bombardment of a solid 89Y target on acyclotron.15,16 In order to produce an aqueous 89Zr4+ speciessuitable for radiolabeling reactions, the solid target is rstdissolved with 6 M HCl. Yet this process produces aqueous89Zr4+ and 89Y3+ species that must be separated. To this end, theHCl solution is run through a hydroxamate resin that has highaffinity for 89Zr4+ and very low affinity for 89Y3+, thus completelysequestering the 89Zr4+ cations while allowing the 89Y3+ cationsto pass through. Finally, 89Zr4+ is removed from thehydroxamate resin using an eluent of oxalic acid, producing apuried solution of89Zr4+ that can be employed in radiolabelingreactions.

Aqueous Coordination Chemistry of Some CommonRadiometals.Prior to our discussion of metal-based imagingagents, a brief discussion of the underlying aqueouscoordination chemistry of the radiometals is in order. Formore detail, the reader can consult other excellent and moreexhaustive reviews, chiefamongthem a 2010 Chemical Reviewsarticle from Wadas et al.35,11,1721

To begin, four isotopes of copper have been used for PETimaging: 60Cu (t1/2 = 0.4 h; + yield = 93%; E+ = 3.9 and3.0 MeV), 61Cu (t1/2= 3.32 h;

+ yield = 62%;E+ = 1.2 and1.15 MeV), 62Cu (t1/2= 0.16 h;

+yield = 98%;E+= 2.19 MeV),and, most notably, 64Cu (t1/2= 12.7 h;

+ yield = 19%;E+ =0.656 MeV).22 Of course, the chemistry of each is identical. CuII isthe most biologically relevant oxidation state of the metal. Becauseof its electronic structure, the 3d9 cation typically forms square-planar four-coordinate, square-pyramidal or trigonal-bipyramidalve-coordinate, or octahedral six-coordinate complexes.21,23

However, coordinatively saturating six-coordinate ligands havegenerally proven the chelators with the best in vivo perform-ance.3,24 Cu2+ is neither a particularly hard nor soft cation, so aneffective chelator will almost always feature a mixture of unchargednitrogen donors along with anionic oxygen or sulfur donors inorder to neutralize the 2+ charge of the cation. While DOTA has

been used as a chelator for Cu2+, the Cu-DOTA complex has beenshown to be unstable in vivo, often producing elevated levels ofradiocopper uptake in the liver as a result of demetalation.

Alternatively, other macrocyclic ligands with smaller or cross-bridged cavities , such as NOTA (N3O3) or 4,11-bis-(carboxymethyl)-1,4,8,11-tetrazabicyclo[6.6.2]hexadecane-4,11-di-acetic acid (CB-TE2A; N4O2),have been shown to be excellentchelators of the radiometal.2527 More recently, neutral N6macrocyclic chelators based on sarcophagine scaffolds havebeenshown to be extremely adept at chelating the cation.28,29 Thein vivo reduction of copper from CuII to CuI is possible undersome circumstances. In most cases, this reduction is undesirable,and macrocyclic complexes of CuII generally have reduction

potentials far below the threshold for in vivo reduction. However,in some situations, as we shall see in the Cu-ATSM case study,the reduction of CuII to CuI is a critical step in the biologicalmechanism of the tracer.

Moving on, the only stable oxidation state of gallium in anaqueous environment is 3+. The amphoteric nature of Ga 3+

allows for reactions in acidic and alkaline solutions. At pH > 3,insoluble Ga(OH)3 precipitates out of aqueous solutions, butthis species redissolves to soluble [Ga(OH)4

] at pH > 7.4.30

However, on the radiochemical scale, the formation of insolubleGa(OH)3has been shown to be inconsequential if the overallradiometal concentration is kept below 2.5 106 M.3032

The Ga3+ cation is smaller and harder than the Cu2+ cation andthus typically binds ligands containing multiple anionic oxygen

donors.33,34 While some tetrahedral four-coordinate andsquare-pyramidalve-coordinate complexes are known, octahe-dral six-coordinate complexes are far more common. A varietyof acyclic and macrocyclic chelators have been used with Ga 3+,

with N,N-ethylenedi-L-cysteine (EC; N2S2O2), N,N-bis(2-hydroxybenzyl)ethylenediamine-N,N-diacetic acid (HBED;N2O4), NOTA (N3O3), 1,4,7-trismercaptoethyl-1,4,7-triaza-cyclononane (TACN-TM;N3S3), and DFO (O6) forming par-ticularly stable complexes.3538 As we will discuss later, whileDOTA has been employed quite often with 68Ga3+, the chelatordoes not form a particularly stable complex with the cation.39 In-deed, in this regard, Ga3+ provides an excellent example of theimportance of the cavity size of macrocyclic chelators. While


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NOTA binds the cation exceptionally tightly (logK= 30.1; pM=26.4), the larger cavities of its cousins DOTA (log K =21.3; pM= 15.2) and triethylenetetramine (TETA) (log K=19.7; pM= 14.1) make for far less stable complexes.40,41

Not surprisingly, the chelation chemistry of indium is similarto that of gallium. Like its congener, indiums only stableaqueous oxidation state is 3+.34,42 However, the In3+ cation islarger, has a higher pK

a, andexhibits faster water exchange rates

than its Ga3+ counterpart.34As a result, In3+ is more tolerant ofligands bearing softer thiolate donors and can adopt highercoordination numbers than its group 13 neighbor. In part be-cause of this exibility, In3+ has been shown to form complexes

with a variety of different coordination numbers and geo-metries. These include a ve-coordinate trigonal-bipyramidalcomplex with tris(2-mercaptobenzyl)amine (NS3 + anexogenous ligand), a six-coordinate distorted octahedral com-plex with EC (N2S2O2), a six-coordinate distorted octahedralcomplex with NOTA (N3O3), a seven-coordinate pentagonal-

bipyramidal complex with ethylenediaminetetraacetic acid (EDTA;N2O4 + an exogenous ligand), and an eight-coordinate square-antiprismatic complex with DTPA (N4O4).

36,4348 In practice,however, the vast majority of 111In-labeled bioconjugates haveemployed bifunctional derivatives of DTPA or DOTA.34,42,4952

The biologically relevant oxidation state of yttrium is also 3+.However, the Y3+ cation is much larger than either Ga3+ or In3+,allowing it to form complexes with coordination numbers up to8 or 9. Despite its large size, the Y3+ cation is considered to be ahard Lewis acid, and thus ligands with multiple anionic oxygendonors are usually employed for its chelation. When a ligandoffers fewer than eight donors, exogenous ligands ll thecations coordination sphere, as in its eight-coordinate distorteddodecahedral complex with EDTA (N2O4+ two H2O ligands)and nine-coordinate monocapped square-antiprismatic complex

with 1,4 ,7-tr is(carbamoylmethyl)-1,4,7-triazacyclononane(N3O3+ two H2O ligands).

43,5355 Not surprisingly, however,it has been shown that ligands capable of coordinatively

saturating the metal form more stable complexes. As a result,the two chelators most often used in 86Y-labeled radiopharma-ceuticals both offer eight donors: DOTA forms an eight-coordinate square-antiprismatic complex with a Kd of 22,

while DTPA forms an eight-coordinate monocapped square-antiprismatic complex with a Kdof 24.

49,50,52,5660

As a group IV metal, zirconium exists predominantly in the4+ oxidation state in aqueous solution. The aqueous chemistryof the Zr(H2O)x species can be quite complex, with bothspeciation between various mononuclear and polynuclear statesand solubility highly dependent on the pH.6163With regard tochelation chemistry, however, things simplify somewhat. Thecation is relatively large, and its high charge makes it a very hardLewis acid. As a result, Zr4+ displays a very strong preference for

ligands offering anionic oxygen donors in high coordinationnumbers. For example, Zr4+ has been shown to makeoctadentate, dodecahedral complexes with the well-knownchelators DTPA (N3O5), EDTA (N2O4 + two H2O ligands),and DOTA (N4O4).

64,65 Interestingly, however, while thethermodynamic stability constants for both Zr-EDTA (29)and Zr-DTPA (36) have been shown to be quite high, thepoor kinetic stability of these complexes has rendered themunsuitable for use in vivo.53,58,66 Instead, the vast majority of, ifnot all, published 89Zr-labeled radiotracers have employed DFOas the chelator.6770 DFO is an acyclic siderophore-derivedmolecule that binds 89Zr4+ using three hydroxamate groups,thus providing three neutral and three anionic oxygen ligands.

To date, neither a solid state nor an NMR structure has beendetermined for Zr-DFO, although density functional theory(DFT) calculations suggest that a seven- or eight-coordinatecomplex is formed involving exogenous water molecules inaddition to the ligands six oxygen donors.71

Finally, the chemistry of technetium represents a fairlysignicant departure from the radiometals we have discussed so

far. As a group VIIB metal with a neutral electronicconguration of [Kr]4d65s1, the coordination chemistry of99mTc is very complex: a large number of oxidation states (1to 7+) and a wide variety of coordination geometries (square-pyramidal, octahedral, and heptahedral) are possible.7,18,31,7274

This diversity is a double-edged sword: it allows forconstruction of a range of different 99mTc species, but it alsogives rise to ample redox chemistry and chemically labilespecies that complicate the design of imaging agents.75

Upon elution from the generator as tetrahedral 99mTcO4,

99mTc exists in a 7+ state that is not immediately useful forchelation or binding directly tosmall molecules because of itsnegligible chemical reactivity.18 Indeed, there are very fewexamples of the incorporation of TcVII into imaging agents, with99m

Tc-sulfur-colloid (Tc2S7) standing as the only majorexample.31,76 Rather, the vast majority of 99mTc-based imagingagents are prepared using 99mTc in a lower oxidation state. As aresult, a reducing agent or the direct reduction of the metalthrough complexation with hard ligands is necessary in thesynthesis of these probes.7,31,75

Not surprisingly, the different oxidation states of technetiumhave different coordination chemistries. TcV is a d2 metal centerthat, in aqueous environments, typically forms either ve-coordinate square-pyramidal or six-coordinate octahedralcomplexes around a TcVO core or six-coordinate octahedralcomplexes around a TcVO2 core. Ligands featuring donorsranging from neutral phosphorus and sulfur atoms to anionicoxygen atoms have been employed, although tetradentate

chelators based on mercaptoacetylglycylglycylglycine, diamine-dithiol, or aminoaminedithiol scaffolds have proven mostcommon.77,78 Complexes based on technetium(V) nitridocores and the condensation reaction between the TcVO centerand hydrazinonicotinamide have also been explored asalternative TcV coordination strategies.79,80 Unfortunately,however, much of the work with TcV cores has ultimately led tocomplexes that are unstable or preparations that are toocumbersome for clinical translation. For example, the 99mTcVOcore is relatively common in radiopharmaceuticals, but thesecomplexes are often labile at the trans position or are hydrolyticallyunstable when exposed to physiological environments.31

Low-spin TcIII d4 complexes have also been studied asalternatives to TcV-based constructs. The TcIII center has been

shown to make both six- and seven-coordinate complexeswithligands featuring a variety of different donor types.81,82

However, the relatively harsh reducing conditions currentlyemployed to form TcIII from pertechnetate represent asignicant obstacle to its routine use.

Recently, many of the most successful developments havecentered on 99mTcI, particularly complexes based on thekinetically inert, low-spin [99mTc(CO)3]

+ d6 core.83,84 Water-soluble [99mTc(CO)3(H2O)3]

+ can be prepared easily from99mTc-pertechnetate under reducing conditions, and the H2Oligands are easily exchanged with various types of ligands,including tris(pyrazoyl)methane derivatives and click-chemistry-derived scaffolds.8590 The lipophilicity of [Tc(CO)3]

+ remains


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somewhat of a concern, however. TcI is a relatively soft cation,and ligands bearing softer donors tend to increase thelipophilicity of the complex further. Thus, chelation systemsmust be chosen carefully in order to strike a suitable balance

between stability and lipophilicity.Regardless of the identity of the metal, synthesis on the

radiochemical scale has a few critical features that set it apart

from the macroscale synthesis of

cold

complexes. The limitedamount of time allowed for synthesis and purication is themost obvious difference, because reaction and puricationconditions must often be designed with the half-life of theradionuclide in mind. A less apparent difference is the strikinglylow absolute concentration of radiometals in most radiolabelingreactions. Generally, the concentration of radiometal is at least 3(and often more) orders of magnitude lower than that of anyother reactants in a radiochemical reaction. This contrastsdramatically with the excess of metal typically employed inmacroscale reactions that aim to achieve the best possiblechemical yield. For this reason, during radiosynthesis reactions,any potential contaminants, particularly metals that may compete

with the radiometal of interest, become a major concern.

Design and Structure of Radiometal-Based ImagingAgents. From a design perspective, radiometalated imagingagents can be grouped into three classes: small metalcomplexes, chelator-based conjugates, and colloids. Small-metal-complex radiotracers are the most structurally straightfor-

ward class, comprised of two essential parts: a centralradiometal and a set of coordinating ligands. These agentsrepresent the purest points of intersection between inorganicchemistry and nuclear imaging, for the metal complexesthemselves are solely responsible for in vivo targeting, uptake,and retention. A number of small-metal-complex PET andSPECT imaging agents have had a signicant impact in theclinic, including 99mTc-bisphosphonates for bone imaging,99mTc-sestamibi for myocardial perfusion imaging, and 64Cu-

PTSM for blood perfusion imaging.6

Chelator-based conjugates, on the other hand, have fourparts: a targeting vector, a radiometal, a chelator, and a linkerconnecting the chelator and targeting vector.4,5,7 The targeting

vector is typically a biomolecule such as a peptide, protein, orantibody. However, synthetic vectors such as nanoparticles andliposomes have come into vogue in recent years. The selectionof a radiometal is governed by both the imaging modality andthe biological half-life of the targeting vector. The most pop-ular radiometals for SPECT imaging are 111In and 99mTc, andthe most popular radiometals for PET imaging are 68Ga, 64Cu,86Y, and 89Zr. However, a variety of other metallic radioisotopesincluding gallium-67 (67Ga), copper-60 (60Cu), titanium-45(45Ti), and technetium-94m (94mTc) have also been produced

and used. Once an imaging modality has been chosen,matching the radioactive half-life of the isotope to the biologicalhalf-life of the biomolecule is critical. For example, 68Ga and99mTc would not be ideal choices for labeling antibodies

because the radionuclides would decay signicantly before theantibody reaches its optimal concentration at the target.Conversely, neither 89Zr nor 111In would be the best choicefor labeling a short peptide because their multiday half-lives

would far exceed the residence time of the peptidic agent.The job of the chelatorinterestingly, from the Greek

(che le

) meaning clawis simple: form a kinetically inert andthermodynamically stable complex with the radiometal in orderto prevent its inadvertent release in vivo. Radiometal chelators

fall into two structural classes: macrocylic and acyclic chelators.While macrocyclic chelators typically offer greater thermody-namic stability, acyclic chelators usually have faster rates ofmetal binding.18 Generally, transition-metal chelators offer atleast four (and usually six or more) coordinating atoms arrayedin a conguration that suits the preferred geometry of the metalin question. As we have discussed above, different metals preferdifferent chelators, and therefore the choice of chelator isdictated by the identity of the radiometal.

For the linkage between the chelator and targeting vector,the only requirements are that the link must be stable underphysiological conditions and must not signicantly compromisethe binding strength or specicity of the vector. The specicchemical nature of the conjugation method is dependent on

both the type of vector and the availability of bifunctionalvariants of the desired chelator. For vectors with free thiolgroups, the reaction between a thiol and a maleimide hasproven a popular route; for vectors with free amine groups, theformation of thiourea bonds using isothiocyanates or peptide

bonds using activated carboxylic acids has been widelyemployed. It is important to remember, however, that theconjugation of a chelator to a vector may alter its ability tocoordinate a given radiometal. For example, conjugating DOTAto a peptide using one of its carboxylate arms leaves only athree-armed DOTA, more properly termed DO3A, forchelation of the radiometal. In light of this, the use of

bifunctional chelators with pendant conjugation handles, e.g.,[S-2-(aminobenzyl)1,4,7-triazacyclononane-1,4,7-triacetic acid(p-NH2Bn-NOTA) or N-(2-aminoethyl)-trans-1,2-diaminocy-clohexane-N,N,N-pentaacetic acid (CHX-A-DTPA), is oftenpreferable.

The third class of radiometal-based imaging agents, colloids,is the oldest of the three, yet it boasts only one prominentexample: the family of 99mTc-radiocolloids.9193 Nevertheless,99mTc-radiocolloids have had a profound impact on the clinicalimaging of the reticuloendothelial system (RES). Broadlyspeaking, colloids are particles that range in size from 1 nm to 4m. In the body, they are typically removed from circulation viaphagocytosis, a process especially active in macrophages.Consequently, when radiolabeled, they can be used to imagetissues with high concentrations of macrophages, such as theliver, spleen, bone marrow, and lymph nodes. As a result,99mTc-radiocolloids have proven especially important in theimaging of the lymphatic system in oncology. 99mTc-colloids ofa wide range of diameters have been created using a variety ofmaterials, including denatured human albumin, sulfur, anti-mony, and stannous phytate. Somewhat surprisingly, theliterature contains very few allusions to the use of otherradiometals in colloidal imaging agents.94 A more detaileddiscussion of the synthesis and application of99mTc-colloids can

be found in the last of the ve case studies.

CASE STUDIES

In the following pages, our hope is to use ve metal-based radio-pharmaceuticals64Cu-ATSM, 68Ga-DOTATOC, 89Zr-transferrin,99mTc-sestamibi, and 99mTc-colloidas lenses to illustrate thefundamental role of inorganic chemistry in the development of both

well-established and next-generation nuclear imaging agents. Takentogether, we believe that these vignettes will provide both a soundoverview of the different ways inorganic chemistry inuencesradiopharmaceuticals and an arena for the celebration of the integralcontributions of inorganic chemistry to nuclear imaging, while


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simultaneously pointing out areas in which inorganic chemistrycould play a role moving forward.

CU-ATSM: TARGETING TUMOR PHENOTYPE WITHA SMALL METAL COMPLEX

The rst PET imaging agent we will discuss is the hypoxia-targeting small-metal-complex copper(II) diacetylbis(N4-methyl-

thiosemicarbazone), more commonly referred to as Cu-ATSM.95

97

Structurally, Cu-ATSM is a relatively simple metal complex: a CuII 3d9

metal center coordinated in a square-planar geometry by twonitrogen atoms and two sulfur atoms of a tetradentate

bis(thiosemicarbazone) ligand (Figure 2A). Cu-ATSM has

been radiolabeled and studied with all four positron-emittingradioisotopes of copper: 60Cu, 61Cu, 62Cu, and 64Cu. Regardlessof the isotope, however, Cu-ATSM is prepared through thesimple incubation of CuCl2 and the free ligand H2ATSM andpuried using a reverse-phase C18cartridge.

Background and In Vitro Characterization. As its namesuggests, the term hypoxiadescribes the pathological condition in

which a tissue is deprived of normal physiological levels of oxygen.Under normal conditions, the mean arterial partial pressure of

oxygen (pO2) is 70

100 mmHg. In cancerous tissues, however, theerratic and disorganized vasculature of the growing tumor oftenresults in dramatic reductions inpO2in many cases to


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the paltry 3 min of wild-type SST).133,134 A variant calledoctreotide, a cyclic octapeptide with two D-amino acid residues,

was approved by the Food and Drug Administration (FDA) asa therapeuticagent for patients with hormone-secreting tumors(Figure4B).132,135,136 The remarkable tumor-targeting proper-ties of this peptide spurred signicant interest in using SSTanalogues as vehicles for the delivery of both therapeutic anddiagnostic radionuclides. In response, an 111In-labeled variant ofoctreotide111In-DTPA-octreotide (Figure 4C)was devel-oped and ultimately became the clinical standard radiotracer forSPECT imaging of SSTr-positive malignancies.52,137,138

From 111In-Octreoscan to 68Ga-DOTATOC. Over time,optimization of SST constructs to improve in vivo stabilityresulted in a shift in the chelators from DTPA to what was atthe time a novel macrocyclic chelator: DOTA.139 Indeed,111In-labeled conjugates employing DOTA displayed diminishedrenal toxicity and increased tumor accumulation compared to111In-DTPA-octreotidewhile retaining similar overall pharma-cokinetic properties.51,139 Another trait of DOTA viewed asadvantageous was its versatility with other radiometals,particularly positron-emitting 68Ga. At the time, interest inusing PET to image SSTr-positive malignances was on the rise,and the short half-life of 68Ga (67.7 min; + = 89%) makes it anearly ideal match for peptides with rapid pharmacokineticproles like octreotide (t1/2 100 min).

140142

Solid-state structures demonstrate that Ga3+ forms a fairly

typical octahedral six-coordinate complex with DOTA, with themetal bound to the four nitrogen atoms from the macrocycliccage and two oxygen atoms from the pendant carboxylatearms of DOTA.143 The Ga-DOTA complex exhibits relativelymodest thermodynamic stability constants (log KML 21.33)and pMvalues (15.2). However, over the years, it has exhibitedsomewhat surprising in vivo stability in many applications.41,46,144

Despite these limitationsindeed, almost by defaultDOTAbecame the chelator of choice for 68Ga in SSTr imaging. It hasproven a successful, although slightlyawed, marriage.

Production of68Ga3+ and Synthesis of68Ga-DOTATOC.68Ga is produced via electron capture decay (EC) of its parentradionuclide 68Ge (t1/2= 270.95 days) and can thus be producedusing a compact, cost-effective, and convenient generator

system.145

147 In a 68Ge/68Ga generator, 68Ge is immobilized ina matrix of alumina, TiO2, or SnO2, and upon decay,

68Ga3+ isformed, which has a lower affinity for the solid support than itsparent and thus can be eluted in dilute acid.145,148

The 68-min half-life of 68Ga makes radiosyntheses challeng-ing. Most radiolabeling reactions for DOTATOC employconditions involving heating to 90100 C for 515 min inacetate buffers or HEPES (0.10.5 M) with pH ranges of3.05.5 to drive the complexation reaction.147,149152

However, these conditions have required further optimizationbecause of the rapid decay of the radioisotope.131,149,150,152

Ignoring the time required for purication, a simple 15-minincubation will result in the decay of 14% of the initial

radioactivity.131,152 To circumvent this problem, Velikyan et al.explored different reaction conditions and observed that, in amicrowave-assisted reaction, 68Ga-DOTATOC can be synthe-sized in extremely high yields exceptionally rapidly: a 1-minincubation at 90 C.150

As a result of 68Gas simple production, advantageous half-life, and generally favorable coordination chemistry with

DOTA, 68

Ga-DOTA-conjugated peptides have been one ofthe most common classes of imaging agents reported recently.68Ga-DOTATOC, in particular, has stood as the preeminentpeptide-based, receptor-targeted PET imaging probe.153 In fact,compared to 111In-DTPA-octreotide, 68Ga-DOTATOC hasdemonstrated superior resolution as well as a greater ability toprecisely measure the tumor receptor density in a number ofpreclinical and clinical studies (Figure5).149,154156 Furthermore,

68Ga-DOTATOC has proven effective for the selectionof patientslikely to respond to radiotherapy with 90Y-DOTATOC.157

The Future of 68Ga-Labeled Peptides: Moving BeyondGood Enough. A kinetically inert and thermodynamicallystable radiometalchelate complex is crucial to the success of achelator-based radiopharmaceutical. Indeed, in vivo stability isimperative because demetalation of the radiometal can lowertumor-to-nontarget organ activity ratios, thereby decreasingcontrast in imaging applications and increasing toxicity.68Ga-labeled peptides are no exception. For Ga3+, trans-chelation and ligand exchange to serum apotransferrin is aparticular concern because this ubiquitous protein has beenshown to possess two sites with a strong affinity for Ga3+

Figure 4. (A) Somatostatin; (B) octreotide; (C) 111In-DTPA-labeled

octreotide.

Figure 5. (A) Small-animal PET images using 68Ga-DOTATOCshowing tumor delineation in a mouse bearing subcutaneousxenografts that express different levels of SSTr2 (CT = C6-SSTr2,JT = Jurkat-SSTr2, and UT = U87-SSTr2; SSTr2 expression CT > UT > JT).This research was originally published in the Journal of Nuclear

Medicine(see ref131). Copyright 2011 Society of Nuclear Medicineand Molecular Imaging, Inc. (B) 68Ga-DOTATOC PET images (left,anterior view; right, posterior view) of a patient with SSTr(+)abdominal lymph nodes (arrows). (C) SPECT images of111In-DTPA-octreotide in the same patient displaying reduced resolution. Adaptedand reprinted with kind permission from ref 156. Copyright 2007Springer Science + Business Media.


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(log K1Gatransferrin = 20.3; log K2Gatransferrin = 19.3).30,158

Ultimately, while DOTA has been used to great effect with68Ga3+, particularly in 68Ga-DOTATOC, it is not an idealchelator for the radiometal. Simply put, DOTA is merely goodenough. The central cavity is too large to provide optimalthermodynamic stability, and its chelation kinetics are somewhatsluggish. This, in turn, requires radiolabeling reactions with either

long reaction times or elevated incubation temperatures,conditions that are incompatible with both the half-life of theradionuclide and the sensitivity of some biomolecules.

In response to the limitations of DOTA, a number of novelchelators for Ga3+ have emerged. Likely, the best knownexample in the polyazamacrocycle family is NOTA (Figure6A).

NOTA is an exceptional chelator of68Ga3+.31,159,160 The crystalstructure of the Ga-NOTA complex reveals a distortedoctahedral geometry with Ga3+ bound to the three nitrogenatoms of the ligands annular ringand the three oxygen atomsof the pendant carboxylate arms.159 Stability studies demon-strate that NOTA chelates Ga3+ with a stability constant of log

KML= 30.98, almost 10 ordersof magnitude higher than that ofthe Ga-DOTA complex.45,161 Further, this macrocycle can belabeled with 68Ga3+ at room temperature with a reaction time

of 10 min at pH 3.05.5, affording more mild conditions forheat-sensitive biological vectors.147,162 The versatility of thischelator was recently illustrated using a radiolabeled octreotideconjugate. A peptide conjugated to the NOTA variant 1,4,7-triazacyclononane-1-glutaric acid-4,7-acetic acid demonstrated ahigh affinity for both 111In and 68/67Ga, and the resultingSPECT and PET radiotracers exhibited enhanced SSTr2targeting with improved pharmacokinetics and enhancedin vivo metabolic stability.163

Another macrocyclic ligand, N,N,N-triazacyclononanetrisubstituted with methyl(2-carboxyethyl)phosphinic acidpendant arms (TRAP-Pr; Figure 6B), has also shownconsiderable promise for 68Ga3+ because it can complex the

cation more selectively than NOTA or DOTA.164,165 TRAP-Prhas also been shown to bind Ga3+ at considerably acidic pHlevels and with satisfactory kinetic inertness in a range of pHenvironments.166 This trait is crucial in radiopharmaceutical kitformulations because it eliminates stabilizing ligands and cum-

bersome pH adjustments. Furthermore, the stability constant ofTRAP-Pr with Ga3+ (logKML= 26.6), while lower than that of

NOTA, is several orders of magnitude higher than both DOTAand apotransferrin.A third intriguing class of Ga3+ chelators based on the acyclic

pyridinecarboxylate-derived scaffold breaks the macrocyclic moldestablished by DOTA, NOTA, and TRAP. The ligand H2dedpa(log KML = 28.11; Figure6C), for example, achieved the highestspecic activity against other chelators labeled under roomtemperature conditions and with no prior purication of the68Ga eluent.160 Further, in an apotransferrin stability challenge,68Ga-dedpa remained intact after 2 h, a result comparable to that of68Ga-NOTA. Finally, CP256 (Figure6D) is a tris(hydroxypyridinone)ligand that rapidly (


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lessons conferred from over half a century of research on thisbiomolecule.

Biological Background and Previous Attempts atNuclear Imaging with Transferrin. In order to accom-modate their elevated Fe3+ demand, cancerous tissues generallyexpress higher levels of TFRC than normal tissues. As a result,extensive interest has been dedicated to targeting TFRC fordiagnostic and therapeutic applications.170172 Indeed, one ofthe rst milestones in nuclear medicine was the discovery that67Ga3+ (a radiometal that rapidly binds Tf in serum to targetTRFC) can clearly distinguish the presence or absence oflymphomapostchemotherapy where computed tomography isambiguous.173176 On this basis, several groups have attemptedto modernize TFRC imaging, and Tf has been labeled with >10different radionuclides.

Some overpowering limitations have stied the translation ofthese radioconjugates for cancer imaging. Although a systematiccomparison of all Tf-based radiotracers in matched tumormodels is missing from the literature, an analysis of publisheddata suggests three discouraging trends: (1) qualitative, low-resolution images representing no obvious improvement over67Ga-citrate; (2) unfavorable radiotracer catabolism in bio-logical uids that prevents maximal tumor contrast; (3) the useof radionuclides with half-lives not well suited to detectingTFRC expression on tumor cells.

Underscoring all of these shortcomings are the choice ofradionuclide and the methodology used to attach it to Tf. Anadmittedly mundane example is how labeling Tf withradionuclides for SPECT (e.g., 99mTc, 111In) produces imageson par with 67Ga-citrate and inferior to those derivedfrom Tfadducts labeled with positron-emitting isotopes.177180 Lessobvious are the nuances associated with how the radiolabelingstrategy can impact image quality. For instance, engaging theendogenous iron binding pocket of Tf with a radiometal(e.g., 67/68/69Ga, 64Cu, 97Ru, 99mTc, 111In, 113mIn) does notnecessarily result in a sufficiently stable conjugate for optimalin vivo imaging of tumors.181191 These ndings are perhaps

best rationalized on the basis of Tfs avidity for Fe3+: anexhaustive survey of transition metals has shown that few bind(or are predicted to bind) to Tf with the affinity of Fe3+.192,193

In this regard, cation exchange with Fe3+ in situ may liberate theradionuclide from Tf, resulting in rapid clearance and/orTf-independent mechanisms of uptake in tissues. In addition, atleast one Fe3+-independent mechanism of Tf-radionuclidedissociation has been proposed.194

There are two noteworthy exceptions to this observation. Tfhas been effectively coupled to iron-52 (52Fe) and 45Ti, twopositron-emitting radionuclides with equal and higher affinityfor Tf compared to Fe3+, respectively.195199 Proof-of-conceptstudies in animals were also encouraging, with disease foci

effectively discriminated from nearby normal tissues. Unfortu-nately, both radionuclides have fairly short half-lives (t1/2 8 hfor 52Fe and 3 h for 45Ti). In light of patient images with mAbscoupled to long-lived isotopes (e.g., 124I and 89Zr), the imagingcommunity now agrees that large biomolecules (>40 kDa) are

best coupled to radioisotopes with half-lives >24 h to allowimagecollection at later time points after injection (i.e., 37 days).200202

Consequently, what was gained in the strength of the interactionbetween Tf and 52Fe or 45Ti would likely be offset by the inabilityto detect the radiotracer beyond several hours after injection.

One sensible response to the aforementioned radiotracercatabolism issue is to radiolabel Tf directly via covalentmodication of amino acid side chains. Two halogenated

Tf adducts have been prepared by exploiting this strategy(18F and 131I).203205 As in the argument against radiolabelingTf with 52Fe and 45Ti for tumor imaging, the 18F-Tf adduct can

be cautiously dismissed on the basis of the short half-life of18F(t1/2 90 min). Conversely,

131I has an appropriately long half-life (t1/2 8 days), but iodination of tyrosine side chains on Tfinvites the opportunity for rapid radiotracer catabolism in vivo

via a different mechanism. Because Tf is internalized by a cellafter binding TFRC, it is, in part, subject to proteasomaldegradation, after which free radioiodotyrosine (or freeradioiodide) can be expelled. This property is not general toall radionuclides, and the community now understands thatresidualizing radionuclides confer better tumor contrast severaldays postinjection.206 This point was shown most elegantly in arecent report comparing 76Br- and 89Zr-labeled METmAb(onartuzumab), an internalizing antibody to the receptortyrosine kinase MET.207 In this study, residualized 89Zrproduced more persistent images of tumors in mice comparedto the nonresidualizing radioisotope bromine-76 (76Br).9

On the basis of these observations, the case for revisitingTFRC imaging by radiolabeling Tf with 89Zr becamecompelling for several reasons. Most importantly, the proper-ties of 89Zr are ideally suited for tumor imaging with a large

biomolecule like Tf. This topic has been expertly reviewedelsewhere,208,209 but an abridged account is that 89Zr is cheaplyproduced from 89Y (the only naturally occurring isotope of89Y), itproduces positrons efficiently (23%), and, most importantly of all,its relatively long physical half-life (t1/2 3 days) is well-suited tothe biological half-life of a large biomolecule like transferrin.

89Zr Chelation Chemistry: DFO and Beyond. Thecompanion bioconjugation chemistry for 89Zr is fairlyrobust.11,210 As we have discussed, 89Zr4+ is a highly charged,oxophilic cation that strongly prefers neutral and anionicoxygen donors to nitrogen or sulfur ligands. In recent years, thesiderophore-derived chelator DFO has emerged as the primary

workhorse chelator for 89Zr4+. DFO is an acyclic chelator that

binds 89Zr4+ using three hydroxamate groups that provide threeneutral and three anionic oxygen ligands, which, according toDFT calculations, are accompanied in solution by two watermolecules to form an overal l octadentate coordinationenvironment (Figure 7).71 While a variety of conjugationstrategies have been reported for the attachment of DFO to

biomolecules, the most common by far is the use of anisothiocyanate-modied derivative of DFO to form a thiourealinkage between the chelator and a lysine of the biomolecule inquestion. The subsequent radiolabeling of the DFO-modiedconstructs proceeds very simply and cleanly via incubation with89Zr4+ for 3060 min at a pH of 6.57.5 at room temperature.Reecting the community-wide enthusiasm for this chemistry,15 mAbs have been radiolabeled with 89Zr-DFO and

evaluated in preclinical models, with two rst-in-man studiesreported for 89Zr-U36 and 89Zr-trastuzumab.11,210218

However, we would be remiss if we did not point out thatDFO is not an ideal chelator for 89Zr4+. Admittedly, thethermodynamic and kinetic stabilities of its complex with Zr4+

and its favorable radiolabeling conditions make it an extremelyviable option, well beyond the good enough of DOTA and68Ga3+, but signicant improvements can still be made. Anumber of preclinical in vivo studies with 89Zr-DFO-labeledtracers have shown moderate levels of uptake of radioactivity(typically between 5 and 15% ID/g) in the bone, a result of therelease of the osteophilic 89Zr4+ cation. While similar boneuptake has not been observed in early human clinical trials of


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89Zr-DFO-labeled antibodies, such background uptake has thepotential to be a dosimetry concern for clinicians goingforward. Consequently, a number of different groups, includingours, have been working toward the development of novel,more stable chelators for 89Zr. A common thread in much ofthis work is the creation of octodentate geometries rich inoxygen ligands, thereby reducing solvent accessibility to themetal center and increasing stability. While no reports of new

ligands have appeared in the literature as of the writing of thismanuscript, we believe the next few years will witnesssignicant strides in the development of chelators for Zr4+.

Synthesis and Validation of89Zr-Tf.With these virtues inmind, our group disclosed the rst synthesis and character-ization of 89Zr-Tf in 2012.219 Owing to a prior report showingthat Zr4+ does not stably bind the Fe3+ pockets on Tf, we choseto conjugate DFO to Tf using a bifunctional variant of DFO

bearing an activated succinyl ester.220 The synthesis of DFO-Tfwas achieved in three steps following a protocol that weadapted from the literature.70 89Zr radiolabeling was consis-tently performed to >90% yield, and 89Zr-Tf was puried usingsize-exclusion chromatography. As expected, 89Zr-Tf was stablefor >96 h in serum ex vivo and in vivo and was resistant to

challenge with 10-fold excess Fe3+

in vitro. Its biological half-lifein mice was 118 h.While many groups have developed Tf-based radiotracers

with the intent to detect tumor tissue, we felt that recent datadescribing the mechanisms of TFRC regulation argued morestrongly for using 89Zr-Tf to monitor the treatment response totargeted therapies. Indeed, three highly visible oncogenic eventsregulate TFRC biology. First, TFRC is a target gene of thetranscription factor MYC, an oncogenic driver of manycancers.221223 Second, TFRC is a target gene of the HIF1transcription factor, which is upregulated in many tumors withPI3K pathway activation.224226 Finally, the kinase SRC canregulate clathrin-dependent endocytosis of the Tf-TFRCcomplex.227,228 Each of these oncogenic events are immediately

or distally druggable

with targeted therapies, and given thatpatient response to targeted therapies is notoriously variable orshort-lived, we proposed that 89Zr-Tf could serve as a powerfulpost-therapy response indicator to quickly evaluate tumor

biology.We validated this concept by applying 89Zr-Tf to MYC-driven

prostate cancer models, owing to the clarity of the data linkingMYC activity to TFRC expression. Remarkably, 89Zr-Tfquantitatively measured treatment-induced changes in MYC(and TFRC) expression in MycCaP xenografts.229 Moreover,89Zr-Tf detected spontaneously arising MYC-driven prostatecancer in a transgenic mouse model with prostate-specic MYCoverexpression (Figure 8).230,231 The radiotracer also detected

small foci of aberrant MYC activity in mice with prostaticintraepithelial neoplasia, a clinically validated precursor to prostate

cancer. On the basis of the promise of thesendings, arst-in-manstudy for 89Zr-Tf in patients with newly diagnosed prostate canceris planned at MSKCC, and we are actively investigating the abilityof 89Zr-Tf to measure the pharmacological inhibition of PI3Kpathway constituents and SRC in preclinical cancer models.232,233

Although Tfs historical importance as a catalyst for newresearch in imaging is clear, that no radiotracers have achievedregulatory approval to replace 67Ga-citrate for tumor targetingshould provoke reection among the chemistry and imagingcommunities. Because the shortcomings of experimental Tf-

based radiotracers are generally related to the method of theirpreparation, this narrative speaks convincingly to the impor-tance of sustained communication between inorganic chemists,radiochemists, and biologists to rationally engineer the most

appropriate radiotracer for the task.

99MTC-SESTAMIBI AND 99MTC-COLLOIDS: USINGINORGANIC CHEMISTRY TO CREATE TWO VASTLYDIFFERENT IMAGING AGENTS WITH THE SAMEISOTOPE

Thenal two case studies that we will discuss99mTc-sestamibiand 99mTc-colloidsare based on the -emitting isotope99mTc.31,72,234,235 Although 99mTc was rst identied in 1938,it was not until the 1960s that its potential in the development ofradiopharmaceuticals was fully acknowledged and the rstcommercial generator technology was developed.236,237 Theproduction of99mTc begins with the isolation of molybdenum-99

Figure 8. Representative coronal slices of a coregistered PET/CTshowing the distribution of 89Zr-Tf in a genetically engineered mousewith prostate-specic overexpression of MYC. The animal was 12months old, a time point at which invasive adenocarcinoma haddeveloped. The images were acquired 16 h postinjection of89Zr-Tf. Exvivo analysis conrmed uptake of the radiotracer in malignant tissue.Abbreviations: H = heart; L = liver; B = bladder; PCa = prostatecancer. This research was originally published in Nature Medicine(seeref231). Copyright 2012 Nature Publishing Group, Inc.

Figure 7.(A) Structure of DFO. (B) Simple binding scheme of Zr4+ with DFO. (C) Calculated DFT structure of Zr4+ with DFO. Adapted withpermission from ref11. Copyright 2012 Elsevier Publishing Group, Inc.


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(99Mo), which is most commonly extracted from the ssionproducts of the neutron-irradiated uranium-235 (235U) fuel fromnuclear reactors. After initial processing, 99Mo, in the form ofmolybdate (MoO4

2), is used to generate 99mTc based on theconcept of ion-exchange chromatography. 99MoO4

2 is loaded

onto an acidic alumina column (Al2O3) that is housed inside atransportable, shielded container (technetium cow) fordistribution.238 Upon the decay of 99Mo, 99mTc-pertechnetate(99mTcO4

1) is produced, which, because of its single charge, hasa far lower affinity for alumina. Therefore, when a saline solutionis ushed through the system (milking), 99mTcO4

is eluted,while 99MoO4

2 is retained on the alumina.99Mo decays primarily through a -decay process (87%)

that yields the metastable 99mTc, while a minor decay pathway(13%) results in the formation of technetium-99 (99Tc), aradioactive nuclide that is essentially stable (t1/2 212000

years) and ultimately produces stable ruthenium-99 (99Ru).99mTc, in contrast, possesses a half-life of approximately 6 h anddecays predominantly (>98%) via the emission of 140 keV

-rays that are readily detected by commercially available-cameras for SPECT applications.75

99mTc-sestamibi. The most prolic radiopharmaceuticalagent currently in use is 99m Tc-hexak is( 2-methoxy-2 -methylpropyl)nitrile (99mTc-sestamibi).239 99mTc-sestamibi is afairly simple metal complex composed of a coordinatively inert99mTcI d6 core surrounded by an octahedral array of (2-methoxy-2-methylpropyl)nitrile ligands (Figure 9A). As a

whole, 99mTc-sestamibi is lipophilic and positively charged(1+), characteristics that lead it to accumulate in themitochondria because of its membrane permeability and thenegative charge present on the inner mitochondrial matrix.240

The contribution of the inorganic chemists who developed theseries of compounds that eventually led to the discovery of99m

Tc-sestamibi cannot be overstated. Many iterations ofligands were investigated, and the optimization of the physico-chemical properties conferred by those ligands led to thediscovery of what is currently the most commonly used imagingagent in the world today. With the radiochemistry and nuclearmedicine community constantly pursuing the next greatradiopharmaceutical, 99mTc-sestamibi stands as a shiningexample of how simple chemistry and rational design canproduce an agent that can stand the test of time.

Unlike most TcI complexes that are produced via directlabeling with free ligands, 99mTc-sestamibi is produced via aligand-exchange process using a preformed CuI complex.75 Theprocess begins with the reduction of pertechnetate. To this end,

99mTcO4 is incubated with a tetrakis(2-methoxyisobutylnitrile)-

copper(I) tetrauoroborate ([Cu(MIBI)4]+[BF4]

) complex atelevated temperature. Although the isonitriles themselves may actas reducing agents, other reducing agents are typically also addedin large excess to aid in the reduction process and ensure high

radiochemical yields. The reducing agent employed in commercialkits for the production of 99mTc-sestamibi is stannous chloride

because it is reliable and nontoxic and the Sn2+ to Sn4+ oxidationreaction occurs with ease under the conditions for productionof 99mTc-sestamibi. Additional reagents and buffers are also in-cluded in these kit formulations in order to maximize radio-chemical yield.

99mTc-sestamibi has three principal applications in the clinic.The rst, and most common by far, is in cardiac imaging. As aresult of this mitochondrial localization, the uptake of 99mTc-sestamibi in the myocardium is directly proportional tomyocardial blood ow.241,242 Two sets of images are collectedin a typical 99mTc-sestamibi cardiac SPECT procedure.243 Therst image is taken 11.5 h after the patient is injected with 7

10 mCi of99mTc-sestamibi during exercise of pharmacologicallyinduced stress. The second image is collected roughly 3 h aftertherst and is performed after the patient has been at rest. Thistwo-part test allows physicians to differentiate betweentransient and persistent abnormalities in perfusion in theheart (Figure9B).244

In addition to its widespread use as a myocardial perfusionimaging agent, 99mTc-sestamibi has also been employed incancer imaging, most notably as a second-line diagnostic for

breast cancer but also in preclinical studies of lymphoma,carcinoma, and sarcoma.245248 Not surprisingly, the efficacy ofmany chemotherapeutics is dependent upon both the amountof drug that accumulates in the tumor and the amount of timeit resides there. However, the extent of accumulation and the

rate of wash out are dependent on a variety of complicatedfactors, including blood ow, vascularization, tissue viability,and tumor metabolism. Consequently, the ability of 99mTc-sestamibi to assess perfusion has been harnessed as an indicatorof the ability of a drug to reach its target. Indeed, the increaseduptake and retention of 99mTc-sestamibi in tumors have insome cases been shown to be predictive of response to certainchemotherapies.249 However, it is important to note that thisstory is still developing, because there are many ongoing clinicalstudies that aim to assess the utility of 99mTc-sestamibi as apredictive marker of treatment response.250

A third application of99mTc-sestamibi is parathyroid imaging,in particular the identication and localization of adenomas in

Figure 9.(A) Structure of99mTc-sestamibi. (B) 99mTc-sestamibi SPECT scintigraphy looking at myocardial perfusion. A 54-year-old man with acuteanterior myocardial infarction initially presented with a perfusion defect of the anteroseptal and apical territories of the heart (left), but 4 monthslater, perfusion defect and ischemia were signicantly reduced (right). Adapted and reprinted with kind permission from ref244. Copyright 2010Springer Science + Business Media.


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the thyroid.251253 Studies have shown that about 60% ofhyperfunctioning parathyroids caused by the development of anadenoma will accumulate 99mTc-sestamibi more quickly thannormal parathyroid glands.251253 For this application, SPECTimages are obtained shortly after injection to identify all glands,and images are obtained again after the tracer has been allowedto wash out, allowing for identication of the abnormal glands.

In more recent years, the eld of 99mTc chemistry hasunquestionably witnessed a number of important advances,including developments in 99mTc-tricarbonyl chemistry (includ-ing kit-based formulations), the use of hydrazinonicatinomidefor 99mTcO-based labelings, and the creation of the cleverclick-to-chelateradiolabeling methodology. In this regard, the

work of Alberto and Schibli in Switzerland, Blower in the U.K.,and Jurisson in the U.S. has been particularly impor-tant.19,20,83,84,87,254256 However, despite these successes, it ishard to avoid the impression that progress in the eld as a

whole seems to have slowed somewhat in recent years. Theoverall lack of new complexes, novel chelators, and effective and

biologically stable 99mTc-based bioconjugates highlights theneed for fresh ideas and new avenues of research.

99mTc-colloids.While 99mTc-sestamibi is the product of thecareful manipulation of the chemical properties conferred byligands, the chemistry of 99mTc-colloids is much less well-dened. That said, while the chemistry and biological targets ofcolloids are very different from other classes of radiopharma-ceuticals, their impact on nuclear imaging has been signicant.Indeed, in recent decades, 99mTc-colloids have undergonenumerous iterative developments and have been approved fornumerous applications.

Generally speaking, colloids are particles of a certain sizerange (1 nm to 4 m) that mimic the types of particles found inhuman body uids that are removed by macrophages viaphagocytosis.257 The majority of the macrophage population inhumans is found in the RES, which is comprised of the lymphnodes, bone marrow, liver, and spleen. Sites of infection and

inammation also experience increases in macrophage pop-ulations. In addition to the particle size, a number of otherfeatures determine the biological fate of 99mTc-colloids andtheir potential imaging applications, including their chargeand potential to interact with biological macromolecules.

As a result, a wide variety of formulations have been used tocreate 99mTc-colloids, including 99mTc-tin-colloid (AmerscanHepatate II), 99mTc-rhenium-sulde-colloid (Sulfotec), and99mTc-albumin-microcolloid (Microlite).

Relative to 99mTc-based small metal complexes andbioconjugates, the radiochemistry of 99mTc-colloids variesgreatly, and the self-assembly process, while tunable, issomewhat poorly dened. For example, production of99mTc-tin-uoride-colloids, which have been used to label

leukocytes in vitro, proceeds from small template particlesthat are formed by nucleation from a supersaturated solution ofreagents, including tin(II) uoride, sodium uoride, 99mTc, andpolaxamer 188 (a stabilizing agent).258,259 Those templateparticles then grow from the remaining reactants in thesolution, capturing the technetium atoms within the macro-molecular complex in a self-assembly processthat can be tuned

by physical manipulation of the solution.260 Factors that mayinuence the formation of the colloid include the pH,incubation time, length and degree of agitation, and the shearforce from drawing the solution into a syringe. These factorscan affect the size of the particles, which can, in turn, inuencetheir biological activity.

In contrast, a very different type of radiochemistry is used inthe production of 99mTc-albumin-nanocolloid (Microlite),

which is commonly used for lymphoscintigraphy, bone marrowscintigraphy, and inammation scintigraphy.94 In this case, thealbumin provides a templatefor the radiocolloid, which is notas sensitive to physical manipulation. As a result, 99mTc-albumin-nanocolloid can be produced in more uniform particle

sizes. The synthesis of 99m

Tc-albumin-nanocolloid is achievedby simply reducing 99mTc-pertechnetate with SnCl2 and thenmixing it with a preformed human serum albumin aggregate atroom temperature.

One of the primary uses of99mTc-colloids is lymphoscintigraphy,particularly sentinel lymph node mapping (Figure 10).261263

Sentinel lymph nodes are the rst node or group of nodes thatreceive drainage from a tumor and, as a result, are particularly proneto metastatic spread. When injected at the site of a tumor, 99mTc-colloids will follow the route of lymphatic drainage because they aretoo large to enter the bloodstream and will be consumed by

resident macrophages upon reaching the sentinel lymph node.Once the process is complete, delineation of the lymph nodes viaSPECT imaging is possible. How well a particular colloid localizesat a sentinel lymph node is determined by its size: particles in the200300 nm range typically will be retained in the rst lymphnode, whereas smaller particles tend to migrate farther and morerapidly through the lymphatic system, a characteristic that may bedesirable depending on the procedure.257,262 Biopsy or radio-guidedresection of the sentinel lymph node is often the goal, in which casea colloid is chosen to balance the speed of migration and amount ofphagocytosis.

In the end, although the chemistry of many99mTc-colloids isnot particularly well elucidated, they have become anindispensable radiopharmaceutical in the clinic. That said, the

chemistry and mechanism of these probes warrant furtherinvestigation to prevent the eld from falling into an ill-advisedsense of if it aint broke, dont fix it. Such information shouldallow more precise ne-tuning of the particle size and shape,

which, in turn, will allow for the ab initio development ofparticles that serve more precise applications in the future.

FRONTIERS FOR INORGANIC CHEMISTRY ANDNUCLEAR IMAGING: THE YEARS AHEAD

Up to this point, we have sought predominantly to highlight thetremendous impact of inorganic chemistry on nuclear medicine.However, we also feel it is important to address a number of thechallenges faced by the two elds in the years to come. Indeed,

Figure 10. 99mTc-sulfur-colloid SPECT lymphoscintigraphy. Anterior(A) and right-lateral (B) transmission images obtained 30 min afterinjection of9 9mTc-sulfur-colloid into the left breast show the injectionsite (solid arrow) and focal uptake (dashed arrow) in the sentinel nodein the right axilla. This research was originally published in the Journalof Nuclear Medicine (see ref263). Copyright 2006 Society of NuclearMedicine and Molecular Imaging, Inc.


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we believe that the relationship between inorganic chemistryand nuclear imaging has never been more important. The useof radiometals in nuclear imaging currently lies at an incrediblyimportant juncture, with the number of radiometal-basedimaging agents in clinical trials at an all-time high and 68Ga-DOTATOC on the cusp of FDA approval in the United States.Expectationsand standardshave never been higher, and aconcerted effort between inorganic chemists, radiochemists,and nuclear imaging scientists will be essential in order to movemetal-based radiopharmaceuticals forward. These challengescan be loosely organized into two groups, lying outside andinside the laboratory.

Growth Outside the Laboratory. 1. Broadening theAvailability of Radiometals. The rst obstacle, and one thatlies more in the purview of politicians and administrators thanscientists, is the lack of widespread availability of manyradiometals, a problem reinforced by the worldwide shortageof 99mTc that started just a few years ago and continues today.The American Medical Isotope Production Act signed into lawthis past January will do much to alleviate concerns over 99mTcin the United States. However, both imaging radiometals suchas 64Cu, 89Zr, and 111In and therapeutic radiometals such as177Lu and 225Ac are still only produced in a handful of facilities

worldwide, and this scarcityboth literal because of geo-graphical logistics and practical because of high costshinders

both the preclinical development and clinical utilization ofradiometal-based imaging agents.

2. Building Bridges between Inorganic Chemistry andNuclear Medicine.Second, it is imperative that stronger links

be formed between the inorganic chemistry, radiochemistry,and nuclear imaging communities. One particularly powerful

way to do this is to more fully integrate radiochemistry andnuclear imaging into the inorganic chemistry curricula ofundergraduate and graduate chemistry departments. Inaddition, facilitating radiochemical research in universitychemistry departmentsrather than in medical centers,

where it is now largely stationed

would ensure a generationof radiochemists and molecular imaging researchers that are

well-trained in the fundamentals of inorganic chemistry. Thiswould no doubt require investment, in terms of both facilitiesand personnel. However, the returns, in the form of better-trained students and productive collaborations, would almostcertainly justify the outlays, particularly in light of the emphasiscurrently being placed on translational science by preeminentfunding agencies. Finally, better incorporation of radio-chemistry and nuclear imaging into chemistry conferencescould facilitate the exchange of ideas. For example, while theannual meeting of the American Chemical Society does haveNuclear Chemistry sections, they are often held in satelliteconference spaces. Moving these sections, spatially and

intellectually, into the main ow of the conference wouldrepresent a small (and symbolic) step in the right direction.

On the ip side, the radiochemistry and nuclear imagingcommunities can certainly contribute to forging these relation-ships, too, particularly by seeking out and hiring trainedinorganic chemists to develop new ligand frameworks andimaging agents rather than simply relying on commerciallyavailable products that have proven good enough.In addition,it would behoove the nuclear medicine eld to place a greateremphasis on fundamental chemistry, both inorganic andorganic, at conferences and symposia and in hospital oruniversity seminar series. Exposure of more biomedicallyoriented researchers to the cutting edge of pure chemistry

can often lead to fruitful and productive collaborations. In theend, the eld as a whole will reap the benets of thesestrengthened bonds: stronger collaborations, more uidexchange of ideas, better-trained scientists, and, ultimately,

better imaging agents.In the end, success in addressing these out of the laboratory

issues will dramatically accelerate scientic progress in both thelaboratory and clinic.

Pressing Needs in the Laboratory. Moving into thelaboratory, the next ve years of science at the intersection ofinorganic chemistry and nuclear medicine will be an excitingtime. Progress is needed and will surely be made on a wide

variety of fronts. Yet we feel it is especially important to brieydiscuss three particularly pressing needs, each inspired by a casestudy discussed above.

1. Drive for New Chelators. The 68Ga-DOTATOC casestudy illuminated the critical role of chelator choice in thesuccess of an imaging agent. As we have discussed, selecting achelator for an imaging agent requires a delicate balancing act.The appropriateness of the chelator for the chosen radiometalmust rst beconsidered but so too must the structure of thechelator itself.4 Macrocyclic chelators typically offer greaterstability but often require elevated labeling temperatures, whichmay compromise sensitive biomolecule vectors; conversely,acyclic chelators often label quickly and easily but are generallyslightly less stable in vivo. Thus, it becomes clear that chelatorscombining the thermodynamic stability and kinetic inertness ofmacrocyclic chelators with the facile radiolabeling of acyclicchelators could be tremendously valuable tools. The ultimategoal, of course, is an ideal chelator: one that can be labeledquickly and cleanly at room temperature and forms a complex

with high kinetic and thermodynamic stabilities.In this regard, newer generations of chelators have offered

dramatic improvements over old workhorses like DOTA andDTPA, rst with NOTA, CHX-A-DTPA, and CB-TE2A andmore recently with constructs based on sarcophagine,

triazacyclononane-phosphinic acid, and pyridinecarboxylatescaffolds.29,164,264,265 However, there is still some room forimprovement, particularly in the development of chelators thatoffer both mild labeling conditions and high in vivo stability.Further, while the development of radiometal-specic chelatorshas proven the simplest route, chelators capable of effectivelycoordinating multiple different radioisotopes are particularlydesirable because of their potential use with imaging andtherapy isotopic pairs. New ligands for 89Zr4+ are an especiallypressing need because only one (DFO) has proven effective todate. While DFO stably sequesters 89Zr, an octadentate chelatormay further increase the stability, thereby eliminating therelease of89Zr4+ in vivo and preventing the background uptakein bone observed in many preclinical studies (vide supra).

Inorganic chemists and nuclear imaging scientists share amutual responsibility for the advancement of new chelators. Onthe chemistry side, it is critical that chemists include in theirpublications the data most relevant to nuclear medicine,including stability measurements under physiological con-ditions and comparisons to more established chelatorsperformed under standardized conditions. Further, becausein vivo experiments are not feasible in many chemistry depart-ments, it is essential that chemists either make bifunctional

variants of their chelators available to the nuclear imagingcommunity or, preferably, develop collaborations with nuclearimaging scientists in order to facilitate in vivo experiments andpush their chelators toward the clinic. On the ip side, imaging


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scientists must seek out new, better chelators rather than simplyrely on established ones.

This cooperation is particularly important given theexigencies of clinical regulatory review. The process is invariablyslow and expensive and, as a result, possesses an intrinsicinertia. Once an imaging agent has started down this path, thereis an understandable resistance to change, even if the currentchelator is merely good enough. The 68Ga-DOTATOC storyprovides an excellent example of this phenomenon. This makesthe initial choice of a chelator especially critical, and it is up to

both inorganic chemists and imaging scientists to work togetherto make sure that the best choices are made.

2. Developing Radiotracers Based on Small MetalComplexes. As we have seen in the 64Cu-ATSM and 99mTc-sestamibi case studies, a variety of imaging agents based onsmall metal complexes, also including 99mTc-Myoview and99mTc-Technecard, have been developed and effectivelytranslated to the clinic. However, these agents have typically

been limited to delineating perfusion or hypoxia.18,19 Targetingspecic molecular biomarkers, on the other hand, has remainedthe domain of radiolabeled bioconjugates and small organicmolecules labeled with radiohalogens. This need not be thecase. Metal complexes offer a versatile scaffold for the creationof three-dimensional molecular recognition architecturescapable of specically binding enzymes or antigens. Indeed,

while the ability of metal complexes to selectively target DNAhas been an area of study for decades, recent years have

witnessed a surge of interest in metal complexes as enzymeinhibitors.266,267 Further, the inherent modularity of metalcomplexes could facilitate alterations to both the ligandenvironment and overall charge of the complex in order tooptimize the affinity, selectivity, and in vivo pharmacokineticsand pharmacodynamics.

3. Expanding the Repertoire of Biological Events That CanBe Imaged. The ability of 89Zr-Tf to move beyondconstituitively expressed cancer biomarkers and provide

information about druggable cellular pathways underlines theimportance of expanding and broadening the array of biologicaltargets for imaging. There is a troubling tendency among preclinicalresearchers to overstudy tumor-targeting strategies for whichrobust technologies already exist. One prominent example is theprostate-specic membrane antigen, a prostate cancer-associated

biomarker for which new technologies are continuously publisheddespite the existence of two fully humanized antibodies (7E11 and

J591) and several highly potent and selective small molecules.268Afar more pressing need for the community is the creation of newapproaches to address pathological events that currently cannot beimaged. Listed below are two such examples accompanied by briefexplanations as to how a more dedicated chemical effort might

bring about rapid advances.

Oligonucleotide Imaging.In essence, cancer is a disease ofgenomic instability and transcriptional rewiring, and in thisregard, the most conceptually obvious manner to distinguishmalignant from normal tissue is to exploit these changes.269

The clinical relevance of radiolabeled antisense oligonucleotidesis debated, owing to the generally poor in vivo stability andpharmacokinetics of these molecules. However, antisensenucleotides are not the only molecules capable of selectivelyand specically binding nucleic acids.270 Indeed, the well-established capacity for metal complexes to selectively targetcertain regions of DNA may be an untapped resource forradiotracer development. Along these lines, the modularity ofthe ligand environment of metal complexes could be especially

advantageous because it could be exploited to customize theselectivity of the complexes for sequence-dependent recog-nition.

Post-translational Modications. The aberrant pro-survivaland proliferative signaling endemic to cancer also require thestable upregulation of many post-translational modications to

biomolecules, including protein phosphorylation, proteinubiquitination, and DNA methylation.269 To our knowledge,no groups have successfully developed radiotracers capable ofdistinguishing any post-translational event. As with oligonu-cleotides, leveraging the exquisite selectivity and affinity ofantibodies to target these motifs is controversial because theability of technologies like cell-penetrating peptides (e.g., TAT)to deliver antibodies to cytosolic antigens is still subject todebate.271 In this regard, freely diffusing small molecules, eithermetal complexes or organic molecules, would seem to be themost feasible option, and some encouraging progress in selectivelytargeting post-translational events with small-molecule cageshas

been made within the molecular recognition community.272 Thisprogress would seem to foreshadow a milestone study for both theimaging and radiotherapyelds, pending a more aggressive effortto optimize these reagents for in vivo applications.

CONCLUSIONIn the preceding pages, it was our aim to shed light not only onthe critical role that inorganic chemistry has played in thesynthesis and development of nuclear imaging agents but alsoon the power and promise of radiometal-based PET andSPECT probes in the clinic. The case studies clearly illustratethe structural and functional diversity that radiometals makepossible. These agents run the structural gamut from discretemetal complexes (64Cu-ATSM) to massive macromolecularassemblies (99mTc-colloids) and have been used to effectivelyimage targets ranging from myocardial perfusion (99mTc-sestamibi) to oncogenic biomarkers (68Ga-DOTATOC).Further, while four of the ve case studies described probes

currently used in the clinic, many more, represented here by89Zr-Tf, have shown signicant preclinical promise and lie onthe cusp of translation to the bedside.

Yet our intention here was not simply to celebrate thefruitful intersection of inorganic chemistry and nuclear imaging.

We also hope that this discussion spurs renewed enthusiasm in theinorganic chemistry community for radiometals and nuclearimaging because we believe that the work of inorganic chemists

will be absolutely indispensible as the eld pushes back currentfrontiers. Ultimately, we sincerely believe that only aninterdisciplinary collaboration between inorganic chemists, radio-chemists, imaging scientists, and clinicians will be able to fullyharness the immense potential of radiometals as diagnostic tools.

AUTHOR INFORMATIONCorresponding Author*E-mail: [email protected]. Phone: 1-646-888-3039. Fax:1-646-888-3059.

NotesThe authors declare no competing nancial interest.

REFERENCES(1) Strauss, H. W.; Mariani, G.; Volterrani, D.; Larson, S. M. Nuclear

Oncology: Pathophysiology and Clinical Applications; Springer: NewYork, 2012.

(2) Heller, G. V.; Hendel, R. C. Handbook of Nuclear Cardiology:Cardiac SPECT and Cardiac PET; Springer: New York, 2012.


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ic401607underscoring the influence of inorganic chemistry on nuclear imaging with radiometalsz

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