Current use and future potential of organometallic radiopharmaceuticals

European Journal of Nuclear Medicine Vol. 29, No. 11, November 2002

Abstract. Contrary to common belief, organometalliccompounds exhibit remarkable stability in aerobic andeven diluted aqueous solutions. Technetium-sestamibi(Cardiolite) is one of the most prominent examples ofthis class of compounds routinely used in nuclear medi-cine. This review summarises the recent progress in la-belling of biomolecules with organometallic complexesfor diagnostic and therapeutic application in radiophar-macy and exemplifies in detail developments focussingon organometallic technetium- and rhenium-tricarbonyltechnologies. The value of such technologies has beenrecognised and they have become a valuable alternativeto common labelling methodologies. An increasing num-ber of groups have started to employ an organometallicprecursor for the purpose of radioactive labelling of vari-ous classes of biomolecules, and the advantages and lim-itations of this new technique are compared with those ofother labelling methods. The synthetic access to appro-priate precursors via double-ligand exchange or aqueouscarbonyl kit preparation for routine application is de-scribed. Strategies and examples for the design of appro-priate bifunctional chelating agents for the Tc/Re-tricar-bonyl core are given. The functionalisation of biomole-cules such as tracers for the central nervous system (do-paminergic and serotonergic), tumour affine peptides(somatostatin receptors, neuroreceptors) and tumourbinding single-chain antibody fragments is summarised.Where possible and appropriate, the in vitro and in vivoresults in respect of these examples are compared withthose obtained with classical 99mTc/188Re(V)- and 111In-labelled analogues. The preclinical results show the inmany ways superior characteristics of organometallic la-belling techniques.

Keywords: Organometallic – Bioorganometallic – Tricar-bonyl – Radiopharmaceuticals – Biomolecule

Abbreviations: MIBI, 2-Methoxyisobutylisonitrile –99mTcCO, [99mTc(H2O)3(CO)3]+ – DAT, dopamine trans-porter – PgR, progesterone receptor – EgR, oestrogen re-ceptor – BFCA, bifunctional chelating agent – n.c.a.,non-carrier-added – scFv, single-chain antibody frag-ments – mAb, monoclonal antibody – NET, norepineph-rine transporter – IPT, [N-(3-iodopropen-2-yl)-2β-carbo-methoxy-3β-(4-chlorophenyl) tropane] – β-CFT, 3β-(4-fluorophenyl)-tropane – Ac, acetyl – DTPA, diethylenetriamine penta-acetate – IDA, iminodiacetate – BC,boranocarbonate – HYNIC, hydrazinonicotinic acid

Eur J Nucl Med (2002) 29:1529–1542DOI 10.1007/s00259-002-0900-8

Introduction

Classical organometallic compounds, compounds with atleast one direct transition metal-carbon bond, are extreme-ly rare in biological systems. The only naturally occurringorganometallic compound so far discovered is the vitaminB12 coenzyme, adenosyl cobalamine. Bio-organometallicscience is a recently emerging discipline of potential im-portance for future directions in fields such as immunolo-gy, receptor research and assay development. In this fast-growing area of interdisciplinary research, numerousgroups have recently reported remarkable progress.

Upon being confronted with the term “organometal-lic”, even chemists will associate it with compoundswhich are air and moisture sensitive and insoluble in wa-ter. Contrary to common belief, organometallic com-pounds indeed exhibit remarkable stability in aerobic,aqueous solutions, offering a judicious choice of metalsand ligand systems. As a matter of fact, an increasingnumber of compounds containing organometallic coresare under consideration for medical use.

P. August Schubiger (✉)Department of Applied Bioscience, ETH Zurich, CH-8057 Zurich, Switzerlande-mail: [email protected].: +41-56-3102843, Fax: +41-56-3102849

Review article

Current use and future potential of organometallic radiopharmaceuticalsRoger Schibli1, P. August Schubiger2

1 Center for Radiopharmaceutical Science ETH-PSI-USZ, Paul Scherrer Institute, CH-5232 Villigen PSI, Switzerland2 Department of Applied Bioscience, ETH Zurich, CH-8057 Zurich, Switzerland

Published online: 6 August 2002© Springer-Verlag 2002

It is not only recently that nuclear medicine and ra-diopharmacy have played a pioneering and leading rolein the development of clinically applicable, organometal-lic drugs. One of the most widely used SPET imagingagents in routine nuclear medicine, technetium-99m ses-tamibi (Cardiolite, Fig 1), is a classical organometalliccompound. Originally developed as a myocardial perfu-sion agent [1], it is nowadays also successfully appliedfor tumour imaging and the detection of multidrug resis-tance [2, 3]. In fact, Cardiolite is the first example of anorganometallic complex routinely used in nuclear medi-cine.

Compared with the “first-generation” radiopharma-ceuticals, the prerequisites for a novel, e.g. target-specif-ic, radiopharmaceutical are greater with respect to its bi-ological activity. The compound has to maintain as muchaffinity and selectivity as possible for the selected targetbut not for other organs and tissue.

Since many of the interesting radionuclides and par-ticularly nuclides with potential therapeutic applicationare transition metals, there is a need for appropriate andefficient labelling procedures for various biomolecules.In this context, the “artificial” character of organometal-lic transition metal compounds might create distinct ad-vantages compared with “natural” drugs, which are oftensubject to unwanted rapid metabolism in vivo. Thus, or-ganometallic approaches might offer a valuable alterna-tive to common labelling protocols.

In the chemical literature there is no sharp definitionfor organometallic compounds. Thus, beside “classical”organometallics, transition metal complexes with or-gano-phosphorus, -sulphur or -selenium chelates canalso be regarded as organometallics. Applying thisbroader definition, many other, predominantly phospho-rus- and sulphur-based complexes developed in radio-pharmacy and used in nuclear medicine can be regardedas organometallics.

For the sake of consistency and clarity, this review ar-ticle will mainly focus on recent developments and theapplication of “classical” organometallic compounds of

technetium and rhenium. The productivity in this field inthe past decade merits an overview of the progress andfuture perspectives of this class of compounds in respectto radiopharmaceutical and nuclear medical applications.

Prerequisites for application of organometalliccompounds

The discovery of a new class of radiopharmaceuticalsstands at the beginning of a long development process.Several critical issues have to be addressed to satisfy theclinical requirements. The preparation of such com-pounds has to be simple, preferably in a kit formulation.Furthermore, the synthesis has to be safe (conditionswhich can be handled within a routine clinical environ-ment) and rapid (several minute to a few hours, also de-pending on the half-life of the radiometal), and shouldresult in products of high radiochemical purity (≥90%).Therefore, the principal limitations are the following: (a)any preparation has to be performed in physiological me-dia; (b) no purification of the final product should benecessary; (c) the reaction time should be short. Scrutinyof the chemical literature shows that these prerequisitesobviously made the introduction and use of organometal-lics rather unattractive until recently.

The technetium- and rhenium-tricarbonyl core

During the past few years the emphasis of technetium-and rhenium-based agents has gradually been shiftingfrom inorganic chemistry to biochemistry, focussing onthe nature of the biological group attached to the metalchelate. Many efforts have been undertaken to “hide” thetechnetium core and to adapt it to the characteristics ofthe corresponding biomolecule. It has been hypothesisedthat the smaller the technetium complex, the higher thelikelihood that the biological activity will not be altered.Stabilisation of the metal complex in vivo is another im-portant issue. It is well known that beside the thermody-namic stability of a complex, its kinetic stability or inert-ness is of equal, and sometime greater importance for anapplication in vivo.

It is unlikely that significant improvements in the la-belling of biomolecules with technetium and rheniumcan be expected with classical nitrogen/sulphur/phospho-rus-based ligand systems, because they have been op-timised during the past 20 years. Thus, further advancesare more likely when novel ligand systems, such as HYNIC or lower oxidation states, are explored.

One of the most promising and developed organome-tallic cores for labelling of biomolecules is the techne-tium- and rhenium-tricarbonyl core. The metal centre isin the low oxidation state +I and is therefore chemicallyvery inert. The M(CO)3 core (M = Tc, Re) is very com-pact, with an almost spherical shape. If the octahedral

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Fig. 1. Structure of Tc-sestamibi (Cardiolite), the first clinicallyapplied organometallic compound in nuclear medicine

coordination sphere is “closed” with an appropriate li-gand system, the metal centre will be efficiently protect-ed against further ligand attack or re-oxidation. In con-trast, the “open” quadratic pyramidal structure of Tc(V)-oxo complexes with a tetradentate chelate is character-ised by unprotected sides, which are prone to ligand at-tack and protonation, which leads to decomposition ofthe original complex. The corresponding Re(V) com-plexes are even more affected by these characteristics.This has to date possibly been the greatest hindrance toextended therapeutic studies employing rhenium iso-topes. Furthermore, a qualitative comparison of the tri-carbonyl core with the widely employed complexes oftechnetium and rhenium in the oxidation state +V, suchas Tc(V)-MAG3, reveals a significantly reduced size(Fig. 2).

Schubiger’s group at PSI succeeded in developing anormal pressure preparation and a fully aqueous-basedpreparation of the precursor [M(H2O)3(CO)3]+ (M =99mTc, 99Tc, Re) in high yields and with excellent (ra-dio)chemical purity [4, 5]. The precursor can be obtainedby reduction and carbonylation of pertechnetate by inter-action with borohydrides and atmospheric pressure ofcarbon monoxide. This procedure circumvents the mostcommon starting materials for tricarbonyl compounds,decacarbonyl and halopentacarbonyl. Although the pub-lished preparation of [99mTc(H2O)3(CO)3]+, abbreviated99mTcCO, was suitable for research purposes, it still re-lied on toxic gaseous carbon monoxide. For a commer-cial radiopharmaceutical kit preparation this is the sub-ject of some concern. The use of solid, stable and non-toxic potassium boranocarbonate, K2[H3BCO2], whichreleases CO upon hydrolysis and can reduce Tc(VII) toTc(I) concomitantly, can be regarded as the ultimatebreakthrough permitting broader application of organo-metallic precursors [6]. The kit will soon be made avail-able for research purposes (Fig. 3). The recently optimi-sed preparation of the corresponding rhenium precursor

[188Re(H2O)3(CO)3]+, which promises to be useful forfuture therapeutic applications, differs only slightly interms of reducing agents and yields [7].

Closely related to the M(CO)3 subgroup is thecpM(CO)3 class of molecules, where cp stands for cyclo-

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Fig. 2. Qualitative size comparison of the organometallic precur-sor [Tc(H2O)3(CO)3]+ (left) and Tc-MAG3 (right) based on X-rayanalyses. Purple = technetium, red = oxygen, grey = carbon, blue= nitrogen, yellow = sulphur. Hydrogen atoms are omitted

Fig. 3. First kit for the preparation of [99mTc(H2O)3(CO)3]+ start-ing from pertechnetate in saline [6]

Fig. 4. A Alberto’s method for preparation of organometallic tech-netium-cyclopentadienyl-tricarbonyl derivatives in aqueous mediastarting from pertechnetate [54]. B Wenzel’s method via double-li-gand-transfer reaction in organic media [9]

pentadienyl. The cpM(CO)3 core is highly lipophilic,making it particularly interesting for functionalisation ofsteroids and drugs that have to pass the blood-brain bar-rier. The fact that the cpM(CO)3 (M = Re) moiety can becoupled to biomolecules without affecting the bindingaffinities has been demonstrated [8]. The inherent advan-tage of the cp ligand is the low molecular weight and thestability of the resulting half-sandwich complex. Thefirst synthesis of functionalised cp derivatives and 99mTcwas reported by Wenzel (Fig. 4B) [9]. 99mTcO4

– was re-duced in presence of ferrocene derivatives andMn(CO)5Br. Katzenellenbogen and co-workers have re-cently presented a modification of this “double ligandtransfer” (DLT) reaction [10, 11]. However, the harsh re-action conditions in organic media and the often pooryields preclude practical application. An elegant, fullyaqueous-based preparation of cp99mTc(CO)3 derivativeswas recently published by Alberto and co-workers(Fig. 4A). The key step for the unique preparation in wa-ter is the introduction of electron-withdrawing substitu-ents, enabling deprotonation of the cp-ring at physiologi-cal pH. As a consequence, straightforward functionali-sation of biomolecules as well as labelling with the99mTc(CO)3 core under reasonable conditions and withsatisfactory yields could be achieved for the first time.

Ligand systems for the M(CO)3 core

The M(CO)3 core was not designed as a “stand-alone”radiopharmaceutical like, for example, Tc-sestamibi, butas a precursor for simple labelling procedures and easierand more appropriate functionalisation of biomolecules.Consequently, various bifunctional ligand and chelatingagents (BFCAs) have been designed and tested. Thethree carbonyl and the three vacant coordination sidesare facially arranged around the octahedral metal centre.This has a favourable influence on the size and geometryof the chelating units. The oxidation state +I allows theapplication of a broad variety of donor and acceptor at-oms [12].

In the search for appropriate mono-, bi- and tridentateligand systems, N-heterocycles such as imidazoles, pyri-dines and pyrazoles, amides, carboxylic acids, thio-ethers, thiols, phosphines and combinations thereof haveproven to coordinate efficiently to the tricarbonyl core(Fig. 5). Some of these functionalities also correspond toside chains of amino acids and are, therefore, of particu-lar interest for labelling of peptides and proteins. Egli etal. have investigated the ability of amino acids and ami-no acid fragments to react with the 99mTc-tricarbonylcore [13]. The most important finding was that histidinereacts quantitatively with the organometallic precursor atconcentrations as low as 10–6 M at 75°C. The extraordi-narily high capacity of imidazole to build and stabilise

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Fig. 5. Examples of bidentateand tridentate chelating sys-tems for the functionalisationof biomolecules for labellingwith the technetium- and rheni-um-tricarbonyl core

hydrogen bonds is the major reason for this characteris-tic.

The low oxidation state of the technetium and rheni-um centre also allows the use of “exotic” ligand systemssuch as isonitriles (as in the case of, for instance, MIBI)or cyclopentadienes. Particularly the cps are of great im-portance, since they represent the one of the smallest“tridentate” chelates for the tricarbonyl core.

The high flexibility of the M(CO)3 core regarding ap-propriate ligands and consequently the simple function-alisation of biomolecules with mono- and bidentate che-lating systems is convincing and tempting from a chemi-cal point of view. However, pharmacokinetic experi-ments have unveiled some characteristics and limitationsregarding the minimal denticity of the chelate for opti-mal clearance rates and stability of model complexes invivo [14]. It has been observed that 99mTc-tricarbonylcomplexes which are coordinated with a tridentate che-lating system reveal good stability when challenged inhuman plasma and with excess cysteine, histidine or glu-tathione. These complexes have also shown very goodclearance from the blood pool and all tissue and organswhen tested in BALB/c mice (Fig. 6). In contrast, com-plexes which are only coordinated in a bidentate fashionshow significant aggregation with plasma proteins in vit-ro and in vivo. They are significantly retained in theblood and in the organs of excretion, such as the liver

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Fig. 6. Biodistribution of two examples of tridentate and two ex-amples of bidentate coordinated 99mTc-tricarbonyl complexes inBALB/c mice, 24 h p.i.

Table 1. Selected examples of novel technetium/rhenium-tricarbonyl labelled compounds for potential radiopharmaceutical application

Biomolecule/ligand Target/potential application Ref.

1. Small molecules (<1,000 Da)

Biotin Avidinylated mAb/tumour pre-targeting [17]Glucose Tumours [18, 19]Oestrogen Oestrogen receptor/breast cancer [20, 21, 22, 23, 24, 25, 26]Progesterone Progesterone receptor/breast cancer [27]Tamoxifen Oestrogen receptors/breast cancer [20]WAY 100635 Serotonergic system [53]Piperidine derivatives Sigma receptors on tumours [28, 29, 30]Haloperidol Dopamine D2 receptors [9]Tropane Dopamine receptors [9, 31, 32]Benzazepine Dopamine D1 receptors [33]MIBI Multidrug resistance/renal imaging [34, 35]Etomidate Adrenal cortex/adrenal cortical tumours [36]

2. Medium size molecules (1,000<Da<15,000)

Somatostatin Somatostatin receptors [37, 38]Neurotensin Neurotensin receptors [39, 40, 41]Neuropeptide Y (NPY) NPY receptors [42]Bombesin Bombesin receptors/small-cell lung cancer [43]Bitisatin Glycoprotein IIb/IIIa receptor/thrombus imaging [44]

3. Large molecules (>15,000 Da)

ScFvs Colon cancer/bladder cancer [45, 46]Surfactant protein B Acute respiratory distress syndrome [47]MAb Bladder cancer [48]

and the kidneys. The overall charge and the lipophilicityof the complexes seem to play only a subordinate role inthese effects, to which several factors may contribute.Among these, the major one may be the fact that tricar-bonyl complexes with bidentate ligands still have a va-cant coordination side, occupied by a substitution labilewater molecule or chloride [15, 16]. Complexation offunctional groups of plasma proteins via this coordina-tion side can explain the prolonged retention in the bloodpool and (consequently) in all organs and tissue. Furtherinvestigations are necessary to confirm this assumptionand to reveal the exact mechanism in vivo. Therefore, al-though the synthetic complexity may be greater, it seemsmore favourable to functionalise biomolecules with tri-dentate chelates as this will yield a better pharmacoki-netic behaviour.

Radiopharmaceutical application of organometalliccompounds of technetium and rhenium

The number of potential applications of tricarbonyl tech-nology which are currently being exploited is remarkable(Table 1). The advantages of the tricarbonyl moiety arevaluable for the labelling of both small biomolecules(<1,000 Da) and large proteins (>15,000 Da).

Examples of small molecules labelled with Tc/Re-tricarbonyl

A particular challenge for the new generation of radio-pharmaceuticals is the labelling of small receptor-target-ing molecules with 99mTc. Especially neuroreceptor-tar-geting molecules have been the subject of extensive in-vestigation in recent years [49]. The feasibility and highpotential of such radiopharmaceuticals was demonstratedby 99mTc-TRODAT-1, a tropane analogue [50, 51]. Abalanced lipophilicity and a low molecular weight areprerequisites of such compounds. A crucial point for allreceptor-based radiopharmaceuticals is high specific ac-tivity, necessary in order to avoid receptor saturation andunwanted pharmacological side-effects.

These requirements are often hard to meet withTc(V) labelling techniques. Furthermore, most of thetetradentate ligand systems produce syn/anti-stereoiso-mers, which can differ significantly in their biologicalcharacteristics, particularly in the case of small mole-cules. The high labelling efficiency of the tricarbonylcore, the small overall size and the organometalliccharacter offer valuable means of overcoming existinghurdles. TROTEC-1 (Fig. 7A) represents the first ex-ample of a tropane derivative (β-CFT) with exception-ally high affinity in vitro to cloned human DAT, func-tionalised with a thioether/carbonyl moiety [32]. Func-tionalisation of the tropane ring was accomplished witha 4,7-dithiaoctaonic acid at position 2-β. While this

system does not overcome the problem of stereoisomerformation, it is promising because of the straightfor-ward functionalisation strategy via an ester group, thesmall size and the high lipophilicity of the complex.The rhenium complex is less polar than Tc(V)-oxo mol-ecules and revealed a tenfold enhanced affinity towardsmonoamine transporters compared with native β-CFTor "3+1” complexes derived from IPT [52]. IC50 valuesfor the analogue are 7.3±1.1 nM for NET (β-CFT,834±45 nM) and 71±1.4 nM for 5HTT (β-CFT,759±47 nM). The high affinity was explained by an en-hanced lipophilic interaction of the organometallic corewith the transporter.

In related efforts, Katzenellenbogen and co-workershave recently investigated the potential use of η5-cyclo-pentadienyltricarbonyl rhenium and technetium deriva-tives of tropane for DAT imaging [31]. Functionalisat-ion of the tropane analogues was accomplished via N-8-alkylation of nortropane or substitution at the paraposition of the 3β-phenyl group (Fig. 7A). The doubleligand transfer reaction was applied to insert the metal-tricarbonyl core. A strong dependence of the affinitiesfor all three monoamine transporters (DA, 5-HT andNE) on the position of functionalisation could be dem-onstrated. The N-8-substituted tropanes showed affini-ties in the low nanomolar range, whereas the 3β-substi-tuted systems had negligible affinities. These results

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Fig. 7. Organometallic derivatives of tropane for the DAT (A) andligands for the serotonergic system (B) and their affinity for thetargeted receptor subtype

are in agreement with observations that the effect ofsubstituents at the para position of 3β-phenyl cannot bepredicted with our current knowledge and is not direct-ly related to the introduction of the organometallic la-bel.

Arylpiperazine is among the most thoroughly studiedCNS ligands for the 5-HT1A subclass of serotonergic re-ceptor. 1-(2-Methoxyphenyl)-piperazine, a truncated de-rivative of WAY 100635, has been functionalised withcyclopentadien and bidentate Schiff-base chelates, en-abling labelling with 99mTcCO with specific activities ofup to 30 GBq/µmol (Fig. 7B) [53]. The Schiff base com-plex revealed an IC50 value of 5±2 nM for the 5-HT1A re-ceptor. For 5-HT2A, dopamine D2 receptors and 5-HTand D transporters, the IC50 values were in the micromo-lar range. Thus, the biological affinity and selectivity ofthe native compound could be preserved in the organo-metallic derivatives. The complexes are stable in physio-logical phosphate buffer for at least 24 h at 37°C. For thepreparation of the cp-arylpiperazine derivative in yields>95%, a one-pot, single-step synthesis was described,starting directly from aqueous [99mTcO4]– [54]. This canbe regarded as significant progress towards routine ap-plication of the tricarbonyl technology, and of the cp-li-gand system in particular.

Metal carbonyl complexes of steroids have been thesubject of intensive investigation for some time. Jaouenand co-workers have exploited their use as a “cold” bio-logical probe for immunological assays, called CIMA(carbonyl metal immuno-assay) [55, 56, 57, 58]. Thisgroup and others have recognised at a very early stagethe potential of this class of compounds for the diagnosisand therapy of steroid receptor-positive breast cancer [8,9, 22, 23]. The groups of Johannsen and Katzenellenb-ogen have synthesised various 17β-progesterone and 7α-oestradiol dithioether and cyclopentadien complexes ofrhenium(I)-tricarbonyl (Fig. 8). The bioconjugates weretested in vitro for binding affinity towards PgR and EgR.The relative binding affinities (RBA; oestradiol =100%;

RU5020 =100%) of organometallic oestradiol deriva-tives were found to be only slightly lower (15%–23%RBA at 25°C) than those of “3+1” and “4+1” complexesof rhenium(III/V) (9%–45% RBA at 25°C) [59]. The dif-ferences in RBA can partially be explained by the higherlogPo/w values measured for the organometallic deriva-tives. RBA was also found to depend on the nature of thespacer between the metal chelate and the steroid moiety.Similar observations and tendencies have been reportedfor the progestin complexes [27]. For both examples, theorganometallic cyclopentadienyl-tricarbonyl systemswere superior to the dithioether-tricarbonyl systems interms of RBA for the corresponding receptors. Thismight be attributed to the diastereomeric nature of the di-thioether complexes and the presence of bulky bromineatoms in the metal coordination sphere. Synthesis of thecorresponding 99mTc analogues and biodistribution stud-ies will help to clarify the usefulness of these systems aseffective imaging agents for PR- and ER-positive breastcancer.

Examples of peptides labelled with Tc/Re-tricarbonyl

The most advanced branch where tricarbonyl technologyis extensively tested and applied is in combination withtumour affine peptides. Tricarbonyl technology can bebeneficial in several respects:

1. The labelling efficiency gives rise to high specific ac-tivities.

2. Purification of the labelled peptides is not usuallynecessary.

3. The convenient functionalisation strategy for peptides(applicable on a solid-phase synthesiser) is a furtheradvantage.

In general, small hydrophilic peptides distribute uni-formly and penetrate readily in tissue and clear efficient-ly from the circulation. Radiolabelling can result in im-portant changes in hydrophilicity and charge, with con-sequences for biodistribution and tissue kinetics. A “neu-tral” and “innocent” labelling method is therefore ofgreat importance.

Somatostatin analogues such as octreotide are amongthe most successful and thoroughly studied compoundsfor systemic radiotherapy and diagnosis. 111In-DTPA-D-Phe1-octreotide is clinically established and represents abenchmark for any further development of peptide-basedradiopharmaceuticals. Apart from HYNIC-functionalisedoctreotide analogues [60, 61], the 99mTc-99m labelling ofoctreotide has met with rather limited success to dateowing to the difficulties with disulphide bond reductionin the presence of stannous chloride in direct as well asindirect labelling approaches. Since Tc/Re-tricarbonyldoes not react with the disulphide bridges, the advantageof employing this technology is evident. 0Tyr3octreotate

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Fig. 8. Organometallic derivatives of oestradiol (A) and progester-one (B) and the relative binding affinity compared with native oes-tradiol and RU5020

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Fig. 9. Octreotate derivativesfunctionalised for labellingwith 99mTcCO. Potential metal-coordinating groups are la-belled in bold

Fig. 10. Gamma-camera images of A [99mTc(CO)3-Nα-Ac-His-0Tyr3octreotate]0, B [99mTc(CO)3-His-0Tyr3octreotate]+ and C [99mTc(CO)3-DTPA-0Tyr3octreotate]3– compared with D [111In-DTPA-0Tyr3octreotate]– in male Lewis rats bearing CA20948pancreatic tumours, 4 h p.i. B, Bladder; K, kidneys; L, liver; T,tumour

tivity in the cells was much higher in the case of themultiple anionic charged complexes (60.1%±4.6% reten-tion for [99mTc(CO)3-DTPA-0Tyr3octreotate]3– vs 17.9%±3.4% retention for [99mTc(CO)3-Nα-Ac-His-0Tyr3octreo-tate]0, after 16 h at 37°C). Biodistribution experimentswere performed in male Lewis rats bearing CA20948pancreatic tumours. Specific uptake could be detected inall somatostatin receptor-positive tissues. It could beshown that more negative charged BFCAs provide aslightly greater tumour retention and predominantly uri-nary excretion. Yet, [99mTc(CO)3-Nα-Ac-His-0Tyr3oct-reotate]0 showed excellent tumour-to-blood ratios of16:1 30 min p.i. and 33:1 4 h p.i (Table 2). These dataare comparable with those published for 99mTc-HYNIC-0Tyr3octreotate and other high-affinity somatostatin re-ceptor binding peptides such as 99mTc-P587 and 99mTc-P829 [60, 61, 62]. A tendency towards better in vivoproperties was observed if the peptide had been function-alised with multidentate or tridentate instead of histidine(bidentate chelate) only. This observation is a direct re-flection of the clearance characteristics of tridentate andbidentate coordinated 99mTcCO model complexes men-tioned earlier.

analogues functionalised with various BFCAs have beentested for labelling with 99mTcCO (Fig. 9) [38]. TheBFCAs gave rise to complexes of different overallcharge (+1 to –3). Aromatic BFCAs such as histidine(bidentate chelate) and Nα-Ac-His (tridentate chelate)gave rise to very high specific activities of 110 GBq/µmoland 220 GBq/µmol, respectively, whereas the aliphatictri- and multidentate BFCAs produced specific activitiesof 10–20 GBq/µmol. In vitro binding studies of the dif-ferent 99mTc-tricarbonyl labelled octreotide analogueswith AR42 cells revealed significant differences in fa-vour of the neutral complexes (29.9%±2.4% binding for[99mTc(CO)3-Nα-Ac-His-0Tyr3octreotate]0 vs 7.2%±0.6%binding for [99mTc(CO)3-DTPA-0Tyr3octreotate]3–, after4 h at 37°C). On the other hand, the retention of the ac-

In comparison with 111In-DTPA-0Tyr3octreotate,[99mTc(CO)3-His-0Tyr3octreotate]+, [99mTc(CO)3-Nα-Ac-His-0Tyr3octreotate]0 and [99mTc(CO)3-DTPA-0Tyr3oct-reotate]3– provided good images of the 99mTc-labelledcompound except in the case of [99mTc(CO)3-His-0Tyr3octreotate]+ after 4 h p.i. (Fig. 10).

Neurotensin analogues [NT(8–13)] targeting recep-tors expressed on a variety of carcinomas [63] have beenlabelled with 99mTcCO. Eleven NT(8–13) analogueswere functionalised at the N-terminus with an Nα-Achistidine or a histidine similar to 0Tyr3octreotate [13, 64].The almost quantitative formation of uniform and stableproducts was observed at ligand concentrations of 10–5

to 5×10–5 M at 75°C. Unspecific labelling through otherside chains of the peptides was not observed. Althoughthe organometallic core is virtually incorporated in thepeptide, it does not reduce the binding affinity of thepeptide. The KD values of the corresponding labelledpeptides varied between 0.2 nM and 3 nM [native pep-tide NT(8–13): KD=1 nM] [39, 40, 41]. A phase I clinicalstudy has been launched with 99mTcCO-labelled NT de-rivatives.

In the case of other receptor-avid peptides and pro-teins which express an endogenous histidine, such asbombesin or neuropeptide Y, the pronounced avidity ofthe tricarbonyl core for histidine can create problemswith unspecific binding. Pre-labelling procedures can beused to circumvent these problems but this procedure iscumbersome [42]. Garcia-Garayoa et al. could show thatsite-directed post-labelling of bombesin with 99mTcCO ispossible by introduction of an Nα-Ac-histidine (triden-tate chelator) at the N-terminus of the peptide [43]. As aresult, a single, stable species was formed and unspecificlabelling was negligible.

Examples of large proteins labelled with Tc/Re-tricarbonyl

Single-chain antibody fragments (scFvs) have the poten-tial for tumour targeting, since they yield high tumour-to-background ratios at early time points. As scFvs be-come available from combinatorial libraries, a general,efficient and straightforward radiolabelling methodwould be desirable to exploit this technology. Unfortu-nately, there has been no convenient 99mTc labellingtechnique for scFvs. Conventional labelling strategies re-ly on the presence of free sulphuryl groups (via cys-teine), which have to be introduced chemically or geneti-cally. The free cysteines present a problem for routineproduction and storage owing to interference with disul-phide bridges of the scFvs, which favours misfolding ofthe protein. Other procedures use reduced disulphidebridges for “direct labelling” of the protein. As a resultthe biological activity is often lost.

The group at the Paul Scherrer Institute and Plückthun et al. have successfully developed a standard

1537


Tab

le2.

Com

para

tive

bio

dist

ribu

tion

(%

ID

/g)o

f va

riou

s 0 T

yr3 o

ctre

otat

e de

riva

tive

s fu

ncti

onal

ised

for

labe

llin

g w

ith

99m

Tc-t

rica

rbon

yl a

nd 1

11In

-DT

PA-0

Tyr3

octr

eota

te in

mal

e L

ewis

rats

bea

ring

CA

2094

8 pa

ncre

atic

tum

ours

. For

str

uctu

re o

f B

FC

As

see

Fig

.9

[BF

CA

]-0 T

yr3 o

ctre

otat

e[N

α-A

c-H

is]

[His

][D

TPA

][i

soD

TPA

][I

DA

][1

11In

-DT

PA]

Tim

e p.

i./ti

ssue

30m

in4

h30

min

4h

30m

in4

h30

min

4h

30m

in4

h60

min

4h

Blo

od0.

2±0.

00.

03±

0.0

3.2±

0.1

0.3±

0.0

0.4±

0.0

0.2±

0.0

0.4±

0.1

0.1±

0.0

0.2±

0.0

0.03

±0.

00.

1±0.

00.

01±

0.0

Liv

er0.

5±0.

10.

2±0.

117

.4±

2.4

2.8±

0.2

0.6±

0.3

0.5±

0.1

0.3±

0.1

0.3±

0.1

0.4±

0.1

0.2±

0.0

n.a.

n.a.

Kid

ney

1.9±

0.2

0.7±

0.1

6.1±

0.7

3.2±

1.2

5.3±

1.0

5.7±

2.3

2.2±

1.1

3.2±

1.2

2.2±

0.7

1.1±

0.2

2.7±

0.9

2.3±

0.3

Pan

crea

s8.

5±1.

15.

9±0.

92.

4±0.

32.

5±0.

91.

7±0.

62.

0±0.

51.

8±0.

92.

0±0.

75.

7±2.

05.

8±1.

86.

8±1.

25.

7±1.

4T

umou

r3.

2±0.

41±

0.2

3.5±

0.3

2.2±

0.4

1.1±

0.1

1.2±

0.2

1.1±

0.1

1.2±

0.4

2.7±

0.3

1.1±

0.2

2.7±

0.4

2.3±

0.7

Tum

our/

bloo

d16

331

72.

55

310

1536

2723

0T

umou

r/li

ver

6.5

50.

20.

72

29

47.

56.

5–

–T

umou

r/ki

dney

1.6

1.5

0.6

0.7

0.2

0.2

0.5

0.4

1.2

11

1

labelling protocol employing the 99mTcCO precursor forscFvs and “mini-antibodies” (bi- and trivalent con-structs of scFvs) carrying an N- or a C-terminal His-tag(Fig. 11) [45, 46, 65]. The method is particularly ele-

gant because the His-tag is genetically expressed forease of purification of the protein on a nickel affinitycolumn. Mixing of the scFvs together with 99mTcCO inbuffer at 37°C for 15 min results in >90% incorporationof the total activity (Fig. 12). No protein aggregationcould be detected as determined by size-exclusion chro-matography. With this gentle procedure specific activi-ties of up to 3.3 GBq/mg protein could be achieved.Displacement experiments in the presence of 100-foldexcess of free histidine resulted in only minor dissocia-tion of activity from the labelled scFvs, which provedthe site specificity and stability of the His-tag labelling.ScFvs bearing no His-tag showed only minor incorpo-ration of the radioactivity (16% incorporation of initialactivity). If the protein had endogenous histidines, lessthan 25% of initial activity was incorporated. These da-ta show that labelling occurs predominantly through theHis-tag. Further experiments showed that three histi-dines in a row are sufficient for stable incorporation of99mTcCO. The in vitro immunoreactivity of a series ofseven different labelled scFvs ranged from 57% to 97%(Table 3). The in vivo stability of labelled scFvs wastested with an anti erbB2/Her2 scFv 4D5 in tumour-bearing nu/nu mice. Analysis of the whole-mouse seraafter 1 h revealed that 75% of the injected activity wasstill migrating with the intact scFvs, whereas the rest ofthe activity was bound to albumin and high-molecular-weight proteins. The tumour localisation was 1.4%

1538


Fig. 11. Section of a computer-generated model of a proteinlabelled with technetium-tricar-bonyl via His4 and His2 of a 5-His-tag

Fig. 12. Incorporation of 99mTcCO into scFvs with a 5-His-tag(◆◆), with endogenous histidine (●) and with no histidine (■) inbuffer at 37°C. After 60 min the labelled protein was challenged witha 100-fold excess of free histidine. Only 1.5% of initial radioactivitywas released in case of the 5-His-tagged scFvs, compared with 29%in the case of scFvs without histidine in the protein sequence

Table 3. Biological activities ofvarious scFvs after 99mTcCO la-belling

ScFv Antigen Method Activity

MOC31 EGP2 Cell binding 90%4D5 c-erbB2 Cell binding 87%M603-H11 Phosphorylcholine Affinity column 97%FITC-E2 FITC-albumin Gel shift 87%M12 Mucin 44-mer peptide, Dynabeads 57%

i.d./g 24 h post injection, giving rise to tumour/bloodratios of approximately 9. Elevated renal uptake andprolonged retention were detected (108.6%±21.7%i.d./g), which presumably represents an inherent clear-ance property of scFvs. The reason why radioiodinatedscFvs do not show such high renal retention of radioac-tivity is the efficient enzymatic dehalogenation in vivo.Waibel et al. have recently shown that lowering the iso-electric point of the protein, by means of succinylationof the lysine side-chain and co-administration of excessL-lysine, can significantly reduce the high renal uptakeof technetium-tricarbonyl labelled scFvs by a factor ofapproximately 2 [66].

Murray et al. have performed an interesting compara-tive study of reduction-mediated and 188ReCO direct la-belling of anti-MUC1 antibodies in vitro [48]. MUC1mucin is up-regulated and abnormally glycosylated inbladder cancer and is a promising target for intravesicalradioimmunotherapy. The preliminary results clearly re-vealed better retention of immunoreactivity of the188ReCO-labelled mAb (80% at 48 h post labelling)compared with the 2-mercaptoethanol-reduced andRe(V)-labelled mAb (<20% at 48 h post labelling). Thissuggests that the carbonyl labelling methodology may bemore appropriate for intravesical radioimmunotherapyusing 188Re.

Certainly more efforts are required to improve the bi-ological characteristics of Tc/ReCO-labelled antibodiesand scFvs in order to permit routine clinical and eventu-ally therapeutic application. However, the stability andefficiency of this gentle labelling technique for scFvsand antibodies is a decisive advantage.

Miscellaneous examples of organometallic radiolabelled biomolecules

Apart from organometallic technetium and rheniumcomplexes, there have been only a few reports on otherorganometallic compounds of radionuclides for potentialradiopharmaceutical use. Jaouen et al. described the in-sertion of ruthenocene at the 17α-postion of oestradiol[25]. The compound showed moderate retention of bind-ing affinity towards the oestrogen receptor (2% com-pared with native oestrogen). For potential therapeuticapplication, it was suggested to use the β-emitting iso-tope 105Ru (β–, 1.917 MeV, t1/2=4.44 h). The use of orga-nometallic [67Ga]dimethylgallium(III) acetylacetonate(67Ga, electron capture, 1.00 MeV, t1/2=3.26 days) formyocardial imaging agent had to be abandoned becauseof the high accumulation of the compound in the liverand the slow clearance from the blood pool. Reversibleassociation of the (CH3)2[67Ga]+ core with red bloodcells was found to be the reason for the disappointing re-sults [67].

Wenzel et al. have described the potential of 97Ru- or103Ru-ruthenocene-glycine derivatives (“Ru-ruppuran”)

as a surrogate for 123I-hippuran [68] (97Ru: γ, 216 keV,t1/2=2.9 days; 103Ru: γ, 763 keV, t1/2=39.4 days). The ab-sence of β-emission, the suitable γ-energy and the longerhalf-life of the ruthenium isotopes compared with io-dine-123 make them attractive. The renal clearance rateof the organometallic derivatives has been comparedwith that of 125I-hippuran in rabbits. A similar renal andplasma clearance pattern was found for 103Ru-ruppuranand 125I-hippuran. Within the first 20 min p.i., 103Ru-ruppuran was eliminated more rapidly than 123I-hipp-uran, while after 20 min p.i. 125I-hippuran cleared better.This tendency was similar for the plasma clearance. Theauthors claim that the absorbed dose in the bladder andthe kidneys would be slightly lower for 97Ru-ruppuranthan for conventional 123I-hippuran.

Conclusion

It is apparent that organometallic compounds are a valu-able and serious alternative to state of the art labellingtechniques. The encouraging results of preclinical andclinical studies with organometallic labelled, tumouraffine peptides and scFv fragments form the basis forfurther investigations. The potential for use of organo-metallic labelling techniques in nuclear medicine willalso depend on the therapeutic success of organometalliccompounds and the availability of appropriate radionucl-ides. In the future, chemists and radiopharmacists will beequally challenged to exploit the aqueous organometallicchemistry of potential radionuclides to develop noveltechniques and compounds for diagnostic and therapeu-tic application.

Acknowledgements. We thank Mary Dyszlewski, Robert Waibel,Ilse Novak-Hofer for their help in preparing the manuscript.

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