Review article
Current use and future potential of organometallic
radiopharmaceuticals
Roger Schibli1, P. August Schubiger2
1 Center for Radiopharmaceutical Science ETH-PSI-USZ, Paul Scherrer Institute, CH-5232 Villigen PSI, Switzerland
2 Department of Applied Bioscience, ETH Zurich, CH-8057 Zurich, Switzerland
Published online: 6 August 2002
© Springer-Verlag 2002
Abstract. Contrary to common belief, organometallic
compounds exhibit remarkable stability in aerobic and
even diluted aqueous solutions. Technetium-sestamibi
(Cardiolite) is one of the most prominent examples of
this class of compounds routinely used in nuclear medi-
cine. This review summarises the recent progress in la-
belling of biomolecules with organometallic complexes
for diagnostic and therapeutic application in radiophar-
macy and exemplifies in detail developments focussing
on organometallic technetium- and rhenium-tricarbonyl
technologies. The value of such technologies has been
recognised and they have become a valuable alternative
to common labelling methodologies. An increasing num-
ber of groups have started to employ an organometallic
precursor 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 of
other labelling methods. The synthetic access to appro-
priate precursors via double-ligand exchange or aqueous
carbonyl 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 tumour
binding single-chain antibody fragments is summarised.
Where possible and appropriate, the in vitro and in vivo
results in respect of these examples are compared with
those obtained with classical 99mTc/188Re(V)- and 111In-
labelled analogues. The preclinical results show the in
many ways superior characteristics of organometallic la-
belling techniques.
P. August Schubiger (✉)
Department of Applied Bioscience, ETH Zurich,
CH-8057 Zurich, Switzerland
e-mail: august.schubiger@psi.ch
Tel.: +41-56-3102843, Fax: +41-56-3102849
European Journal of Nuclear Medicine Vol. 29, No. 11, November 2002
Keywords: Organometallic – Bioorganometallic – Tricar-
bonyl – Radiopharmaceuticals – Biomolecule
Abbreviations: MIBI, 2-Methoxyisobutylisonitrile –
99mTcCO, [99mTc(H O) (CO) ]+ – DAT, dopamine trans-
2 3 3
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, diethylene
triamine penta-acetate – IDA, iminodiacetate – BC,
boranocarbonate – HYNIC, hydrazinonicotinic acid
Eur J Nucl Med (2002) 29:1529–1542
DOI 10.1007/s00259-002-0900-8
Introduction
Classical organometallic compounds, compounds with at
least one direct transition metal-carbon bond, are extreme-
ly rare in biological systems. The only naturally occurring
organometallic compound so far discovered is the vitamin
B12 coenzyme, adenosyl cobalamine. Bio-organometallic
science 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, numerous
groups have recently reported remarkable progress.
Upon being confronted with the term “organometal-
lic”, even chemists will associate it with compounds
which 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 metals
and ligand systems. As a matter of fact, an increasing
number of compounds containing organometallic cores
are under consideration for medical use.
1530
Fig. 1. Structure of Tc-sestamibi (Cardiolite), the first clinically
applied organometallic compound in nuclear medicine
It is not only recently that nuclear medicine and ra-
diopharmacy have played a pioneering and leading role
in the development of clinically applicable, organometal-
lic drugs. One of the most widely used SPET imaging
agents in routine nuclear medicine, technetium-99m ses-
tamibi (Cardiolite, Fig 1), is a classical organometallic
compound. Originally developed as a myocardial perfu-
sion agent [1], it is nowadays also successfully applied
for tumour imaging and the detection of multidrug resis-
tance [2, 3]. In fact, Cardiolite is the first example of an
organometallic 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 much
affinity and selectivity as possible for the selected target
but not for other organs and tissue.
Since many of the interesting radionuclides and par-
ticularly nuclides with potential therapeutic application
are transition metals, there is a need for appropriate and
efficient 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 often
subject 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 definition
for organometallic compounds. Thus, beside “classical”
organometallics, transition metal complexes with or-
gano-phosphorus, -sulphur or -selenium chelates can
also be regarded as organometallics. Applying this
broader definition, many other, predominantly phospho-
rus- and sulphur-based complexes developed in radio-
pharmacy and used in nuclear medicine can be regarded
as organometallics.
For the sake of consistency and clarity, this review ar-
ticle will mainly focus on recent developments and the
application of “classical” organometallic compounds of
European Journal of Nuclear Medicine Vol. 29, No. 11, November 2002
technetium and rhenium. The productivity in this field in
the past decade merits an overview of the progress and
future perspectives of this class of compounds in respect
to radiopharmaceutical and nuclear medical applications.
Prerequisites for application of organometallic
compounds
The discovery of a new class of radiopharmaceuticals
stands at the beginning of a long development process.
Several critical issues have to be addressed to satisfy the
clinical requirements. The preparation of such com-
pounds has to be simple, preferably in a kit formulation.
Furthermore, the synthesis has to be safe (conditions
which 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 should
result 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 be
necessary; (c) the reaction time should be short. Scrutiny
of the chemical literature shows that these prerequisites
obviously 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 shifting
from inorganic chemistry to biochemistry, focussing on
the nature of the biological group attached to the metal
chelate. Many efforts have been undertaken to “hide” the
technetium core and to adapt it to the characteristics of
the corresponding biomolecule. It has been hypothesised
that the smaller the technetium complex, the higher the
likelihood 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 an
application in vivo.
It is unlikely that significant improvements in the la-
belling of biomolecules with technetium and rhenium
can be expected with classical nitrogen/sulphur/phospho-
rus-based ligand systems, because they have been op-
timised during the past 20 years. Thus, further advances
are 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 is
in the low oxidation state +I and is therefore chemically
very inert. The M(CO)3 core (M = Tc, Re) is very com-
pact, with an almost spherical shape. If the octahedral
 1531

Fig. 2. Qualitative size comparison of the organometallic precur-

sor [Tc(H2O)3(CO)3]+ (left) and Tc-MAG3 (right) based on X-ray
analyses. Purple = technetium, red = oxygen, grey = carbon, blue
= nitrogen, yellow = sulphur. Hydrogen atoms are omitted

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- Fig. 3. First kit for the preparation of [99mTc(H2O)3(CO)3]+ start-
trast, the “open” quadratic pyramidal structure of Tc(V)- ing from pertechnetate in saline [6]
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 of
the original complex. The corresponding Re(V) com-
plexes are even more affected by these characteristics.
This has to date possibly been the greatest hindrance to
extended therapeutic studies employing rhenium iso-
topes. Furthermore, a qualitative comparison of the tri-
carbonyl core with the widely employed complexes of
technetium and rhenium in the oxidation state +V, such
as Tc(V)-MAG3, reveals a significantly reduced size
(Fig. 2).
Schubiger’s group at PSI succeeded in developing a
normal pressure preparation and a fully aqueous-based
preparation 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 obtained
by reduction and carbonylation of pertechnetate by inter-
action with borohydrides and atmospheric pressure of
carbon monoxide. This procedure circumvents the most
common starting materials for tricarbonyl compounds,
decacarbonyl and halopentacarbonyl. Although the pub-
lished preparation of [99mTc(H2O)3(CO)3]+, abbreviated
99mTcCO, was suitable for research purposes, it still re-
lied on toxic gaseous carbon monoxide. For a commer- Fig. 4. A Alberto’s method for preparation of organometallic tech-
cial radiopharmaceutical kit preparation this is the sub- netium-cyclopentadienyl-tricarbonyl derivatives in aqueous media
starting from pertechnetate [54]. B Wenzel’s method via double-li-
ject of some concern. The use of solid, stable and non-
gand-transfer reaction in organic media [9]
toxic potassium boranocarbonate, K2[H3BCO2], which
releases CO upon hydrolysis and can reduce Tc(VII) to
Tc(I) concomitantly, can be regarded as the ultimate [188Re(H2O)3(CO)3]+, which promises to be useful for
breakthrough permitting broader application of organo- future therapeutic applications, differs only slightly in
metallic precursors [6]. The kit will soon be made avail- terms of reducing agents and yields [7].
able for research purposes (Fig. 3). The recently optimi- Closely related to the M(CO)3 subgroup is the
sed preparation of the corresponding rhenium precursor cpM(CO)3 class of molecules, where cp stands for cyclo-

European Journal of Nuclear Medicine Vol. 29, No. 11, November 2002
1532
Fig. 5. Examples of bidentate
and tridentate chelating sys-
tems for the functionalisation
of biomolecules for labelling
with the technetium- and rheni-
um-tricarbonyl core
pentadienyl. The cpM(CO)3 core is highly lipophilic,
making it particularly interesting for functionalisation of
steroids and drugs that have to pass the blood-brain bar-
rier. The fact that the cpM(CO)3 (M = Re) moiety can be
coupled to biomolecules without affecting the binding
affinities has been demonstrated [8]. The inherent advan-
tage of the cp ligand is the low molecular weight and the
stability of the resulting half-sandwich complex. The
first synthesis of functionalised cp derivatives and 99mTc
was reported by Wenzel (Fig. 4B) [9]. 99mTcO4– was re-
duced in presence of ferrocene derivatives and
Mn(CO)5Br. Katzenellenbogen and co-workers have re-
cently presented a modification of this “double ligand
transfer” (DLT) reaction [10, 11]. However, the harsh re-
action conditions in organic media and the often poor
yields preclude practical application. An elegant, fully
aqueous-based preparation of cp99mTc(CO)3 derivatives
was 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 the
99mTc(CO) core under reasonable conditions and with
3
satisfactory yields could be achieved for the first time.
European Journal of Nuclear Medicine Vol. 29, No. 11, November 2002
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, but
as a precursor for simple labelling procedures and easier
and more appropriate functionalisation of biomolecules.
Consequently, various bifunctional ligand and chelating
agents (BFCAs) have been designed and tested. The
three carbonyl and the three vacant coordination sides
are facially arranged around the octahedral metal centre.
This has a favourable influence on the size and geometry
of the chelating units. The oxidation state +I allows the
application of a broad variety of donor and acceptor at-
oms [12].
In the search for appropriate mono-, bi- and tridentate
ligand systems, N-heterocycles such as imidazoles, pyri-
dines and pyrazoles, amides, carboxylic acids, thio-
ethers, thiols, phosphines and combinations thereof have
proven to coordinate efficiently to the tricarbonyl core
(Fig. 5). Some of these functionalities also correspond to
side chains of amino acids and are, therefore, of particu-
lar interest for labelling of peptides and proteins. Egli et
al. have investigated the ability of amino acids and ami-
no acid fragments to react with the 99mTc-tricarbonyl
core [13]. The most important finding was that histidine
reacts quantitatively with the organometallic precursor at
concentrations as low as 10–6 M at 75°C. The extraordi-
narily high capacity of imidazole to build and stabilise
 1533

hydrogen bonds is the major reason for this characteris-

Fig. 6. Biodistribution of two examples of tridentate and two ex-
amples of bidentate coordinated 99mTc-tricarbonyl complexes in
BALB/c mice, 24 h p.i.
tic.
The low oxidation state of the technetium and rheni-
um centre also allows the use of “exotic” ligand systems
such 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 limitations
regarding the minimal denticity of the chelate for opti-
mal clearance rates and stability of model complexes in
vivo [14]. It has been observed that 99mTc-tricarbonyl
complexes which are coordinated with a tridentate che-
lating system reveal good stability when challenged in
human plasma and with excess cysteine, histidine or glu-
tathione. These complexes have also shown very good
clearance from the blood pool and all tissue and organs
when tested in BALB/c mice (Fig. 6). In contrast, com-
plexes which are only coordinated in a bidentate fashion
show significant aggregation with plasma proteins in vit-
ro and in vivo. They are significantly retained in the
blood and in the organs of excretion, such as the liver

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]

European Journal of Nuclear Medicine Vol. 29, No. 11, November 2002
1534

and the kidneys. The overall charge and the lipophilicity

of the complexes seem to play only a subordinate role in
these 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 labile
water molecule or chloride [15, 16]. Complexation of
functional groups of plasma proteins via this coordina-
tion side can explain the prolonged retention in the blood
pool and (consequently) in all organs and tissue. Further
investigations are necessary to confirm this assumption
and to reveal the exact mechanism in vivo. Therefore, al-
though the synthetic complexity may be greater, it seems
more favourable to functionalise biomolecules with tri-
dentate chelates as this will yield a better pharmacoki-
netic behaviour.

Radiopharmaceutical application of organometallic

compounds 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 are
valuable 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 high
potential of such radiopharmaceuticals was demonstrated
by 99mTc-TRODAT-1, a tropane analogue [50, 51]. A
balanced lipophilicity and a low molecular weight are
prerequisites of such compounds. A crucial point for all
receptor-based radiopharmaceuticals is high specific ac-
tivity, necessary in order to avoid receptor saturation and
unwanted pharmacological side-effects.
These requirements are often hard to meet with
Tc(V) labelling techniques. Furthermore, most of the
tetradentate ligand systems produce syn/anti-stereoiso-
mers, which can differ significantly in their biological
characteristics, particularly in the case of small mole-
cules. The high labelling efficiency of the tricarbonyl
core, the small overall size and the organometallic
character offer valuable means of overcoming existing
hurdles. 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 with
a 4,7-dithiaoctaonic acid at position 2-β. While this
Fig. 7. Organometallic derivatives of tropane for the DAT (A) and
ligands for the serotonergic system (B) and their affinity for the
targeted receptor subtype
system does not overcome the problem of stereoisomer
formation, it is promising because of the straightfor-
ward functionalisation strategy via an ester group, the
small 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 towards
monoamine transporters compared with native β-CFT
or "3+1” complexes derived from IPT [52]. IC50 values
for 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 core
with the transporter.
In related efforts, Katzenellenbogen and co-workers
have 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 para
position of the 3β-phenyl group (Fig. 7A). The double
ligand transfer reaction was applied to insert the metal-
tricarbonyl core. A strong dependence of the affinities
for all three monoamine transporters (DA, 5-HT and
NE) 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

European Journal of Nuclear Medicine Vol. 29, No. 11, November 2002

Fig. 8. Organometallic derivatives of oestradiol (A) and progester-
one (B) and the relative binding affinity compared with native oes-
tradiol and RU5020
are in agreement with observations that the effect of
substituents at the para position of 3β-phenyl cannot be
predicted with our current knowledge and is not direct-
ly related to the introduction of the organometallic la-
bel.
Arylpiperazine is among the most thoroughly studied
CNS ligands for the 5-HT1A subclass of serotonergic re-
ceptor. 1-(2-Methoxyphenyl)-piperazine, a truncated de-
rivative of WAY 100635, has been functionalised with
cyclopentadien and bidentate Schiff-base chelates, en-
abling labelling with 99mTcCO with specific activities of
up 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-HT
and D transporters, the IC50 values were in the micromo-
lar range. Thus, the biological affinity and selectivity of
the 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 the
preparation of the cp-arylpiperazine derivative in yields
>95%, a one-pot, single-step synthesis was described,
starting directly from aqueous [99mTcO4]– [54]. This can
be 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 the
subject of intensive investigation for some time. Jaouen
and 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]. This
group and others have recognised at a very early stage
the potential of this class of compounds for the diagnosis
and 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 of
rhenium(I)-tricarbonyl (Fig. 8). The bioconjugates were
tested in vitro for binding affinity towards PgR and EgR.
The relative binding affinities (RBA; oestradiol =100%;
European Journal of Nuclear Medicine Vol. 29, No. 11, November 2002
1535
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” complexes
of rhenium(III/V) (9%–45% RBA at 25°C) [59]. The dif-
ferences in RBA can partially be explained by the higher
logPo/w values measured for the organometallic deriva-
tives. RBA was also found to depend on the nature of the
spacer between the metal chelate and the steroid moiety.
Similar observations and tendencies have been reported
for the progestin complexes [27]. For both examples, the
organometallic cyclopentadienyl-tricarbonyl systems
were superior to the dithioether-tricarbonyl systems in
terms of RBA for the corresponding receptors. This
might be attributed to the diastereomeric nature of the di-
thioether complexes and the presence of bulky bromine
atoms in the metal coordination sphere. Synthesis of the
corresponding 99mTc analogues and biodistribution stud-
ies will help to clarify the usefulness of these systems as
effective imaging agents for PR- and ER-positive breast
cancer.
Examples of peptides labelled with Tc/Re-tricarbonyl
The most advanced branch where tricarbonyl technology
is extensively tested and applied is in combination with
tumour affine peptides. Tricarbonyl technology can be
beneficial in several respects:
1. The labelling efficiency gives rise to high specific ac-
tivities.
2. Purification of the labelled peptides is not usually
necessary.
3. The convenient functionalisation strategy for peptides
(applicable on a solid-phase synthesiser) is a further
advantage.
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 of
great importance.
Somatostatin analogues such as octreotide are among
the most successful and thoroughly studied compounds
for systemic radiotherapy and diagnosis. 111In-DTPA-D-
Phe1-octreotide is clinically established and represents a
benchmark for any further development of peptide-based
radiopharmaceuticals. Apart from HYNIC-functionalised
octreotide analogues [60, 61], the 99mTc-99m labelling of
octreotide has met with rather limited success to date
owing to the difficulties with disulphide bond reduction
in the presence of stannous chloride in direct as well as
indirect labelling approaches. Since Tc/Re-tricarbonyl
does not react with the disulphide bridges, the advantage
of employing this technology is evident. 0Tyr3octreotate
1536
Fig. 9. Octreotate derivatives
functionalised for labelling
with 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 CA20948
pancreatic tumours, 4 h p.i. B, Bladder; K, kidneys; L, liver; T,
tumour
analogues functionalised with various BFCAs have been
tested for labelling with 99mTcCO (Fig. 9) [38]. The
BFCAs gave rise to complexes of different overall
charge (+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/µmol
and 220 GBq/µmol, respectively, whereas the aliphatic
tri- and multidentate BFCAs produced specific activities
of 10–20 GBq/µmol. In vitro binding studies of the dif-
ferent 99mTc-tricarbonyl labelled octreotide analogues
with 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–, after
4 h at 37°C). On the other hand, the retention of the ac-
European Journal of Nuclear Medicine Vol. 29, No. 11, November 2002
tivity in the cells was much higher in the case of the
multiple 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 experiments
were performed in male Lewis rats bearing CA20948
pancreatic tumours. Specific uptake could be detected in
all somatostatin receptor-positive tissues. It could be
shown that more negative charged BFCAs provide a
slightly greater tumour retention and predominantly uri-
nary excretion. Yet, [99mTc(CO)3-Nα-Ac-His-0Tyr3oct-
reotate]0 showed excellent tumour-to-blood ratios of
16:1 30 min p.i. and 33:1 4 h p.i (Table 2). These data
are 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 vivo
properties 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 and
bidentate coordinated 99mTcCO model complexes men-
tioned earlier.
 1537

In comparison with 111In-DTPA-0Tyr3octreotate,

Table 2. Comparative biodistribution (% ID/g)of various 0Tyr3octreotate derivatives functionalised for labelling with 99mTc-tricarbonyl and 111In-DTPA-0Tyr3octreotate in male Lewis
[99mTc(CO)3-His-0Tyr3octreotate]+, [99mTc(CO)3-Nα-Ac-

0.01±0.0

2.3±0.3

2.3±0.7
5.7±1.4
His-0Tyr3octreotate]0 and [99mTc(CO)3-DTPA-0Tyr3oct-

n.a.
reotate]3– provided good images of the 99mTc-labelled

230
–
1
4h
compound except in the case of [99mTc(CO)3-

[111In-DTPA]
His-0Tyr3octreotate]+ after 4 h p.i. (Fig. 10).

0.1±0.0

2.7±0.9

2.7±0.4
6.8±1.2
Neurotensin analogues [NT(8–13)] targeting recep-
60 min

n.a.
tors expressed on a variety of carcinomas [63] have been

27
–
1
labelled with 99mTcCO. Eleven NT(8–13) analogues
were functionalised at the N-terminus with an Nα-Ac
0.03±0.0
0.2±0.0
1.1±0.2

1.1±0.2
5.8±1.8
histidine or a histidine similar to 0Tyr3octreotate [13, 64].

6.5
The almost quantitative formation of uniform and stable
4h

36

1
products was observed at ligand concentrations of 10–5
to 5×10–5 M at 75°C. Unspecific labelling through other
0.2±0.0
0.4±0.1
2.2±0.7
5.7±2.0
2.7±0.3
30 min

side chains of the peptides was not observed. Although

[IDA]

7.5
1.2
the organometallic core is virtually incorporated in the
15

peptide, it does not reduce the binding affinity of the

peptide. The KD values of the corresponding labelled
0.1±0.0
0.3±0.1

2.0±0.7
1.2±0.4
3.2±1.2

peptides varied between 0.2 nM and 3 nM [native pep-

0.4
4h

tide NT(8–13): KD=1 nM] [39, 40, 41]. A phase I clinical

10
4

study has been launched with 99mTcCO-labelled NT de-

[isoDTPA]

rivatives.
0.4±0.1
0.3±0.1

1.8±0.9
1.1±0.1
2.2±1.1
30 min

In the case of other receptor-avid peptides and pro-

0.5

teins which express an endogenous histidine, such as

3
9

bombesin or neuropeptide Y, the pronounced avidity of

the tricarbonyl core for histidine can create problems
0.2±0.0
0.5±0.1
5.7±2.3
2.0±0.5
1.2±0.2

with unspecific binding. Pre-labelling procedures can be

0.2
4h

used to circumvent these problems but this procedure is

5
2

cumbersome [42]. Garcia-Garayoa et al. could show that

site-directed post-labelling of bombesin with 99mTcCO is
[DTPA]

0.4±0.0
0.6±0.3

1.7±0.6
1.1±0.1
5.3±1.0
30 min

possible by introduction of an Nα-Ac-histidine (triden-

2.5

0.2

tate chelator) at the N-terminus of the peptide [43]. As a

2

result, a single, stable species was formed and unspecific

rats bearing CA20948 pancreatic tumours. For structure of BFCAs see Fig. 9

labelling was negligible.

0.3±0.0
2.8±0.2

2.5±0.9
2.2±0.4
3.2±1.2

0.7
0.7
4h

Examples of large proteins labelled

3.2±0.1
17.4±2.4
6.1±0.7
2.4±0.3
3.5±0.3

with Tc/Re-tricarbonyl
30 min
[His]

0.2
0.6
1

Single-chain antibody fragments (scFvs) have the poten-

tial for tumour targeting, since they yield high tumour-
0.03±0.0
0.2±0.1
0.7±0.1
5.9±0.9

to-background ratios at early time points. As scFvs be-

1±0.2

1.5

come available from combinatorial libraries, a general,

4h

33
5

efficient and straightforward radiolabelling method

[Nα-Ac-His]

would be desirable to exploit this technology. Unfortu-

0.2±0.0
0.5±0.1
1.9±0.2

3.2±0.4
8.5±1.1
30 min

nately, there has been no convenient 99mTc labelling

6.5
1.6

technique for scFvs. Conventional labelling strategies re-

16

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 routine
[BFCA]-0Tyr3octreotate

production and storage owing to interference with disul-

phide bridges of the scFvs, which favours misfolding of
Tumour/kidney
Time p.i./tissue

the protein. Other procedures use reduced disulphide

Tumour/blood
Tumour/liver

bridges for “direct labelling” of the protein. As a result

Pancreas

the biological activity is often lost.

Tumour
Kidney
Blood
Liver

The group at the Paul Scherrer Institute and

Plückthun et al. have successfully developed a standard

European Journal of Nuclear Medicine Vol. 29, No. 11, November 2002
1538

Table 3. Biological activities of

various scFvs after 99mTcCO la- ScFv Antigen Method Activity
belling
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%

Fig. 11. Section of a computer-

generated model of a protein
labelled with technetium-tricar-
bonyl via His4 and His2 of a 5-
His-tag

gant because the His-tag is genetically expressed for

Fig. 12. Incorporation of 99mTcCO into scFvs with a 5-His-tag
◆ ), with endogenous histidine (●) and with no histidine (■) in
(◆
buffer at 37°C. After 60 min the labelled protein was challenged with
a 100-fold excess of free histidine. Only 1.5% of initial radioactivity
was released in case of the 5-His-tagged scFvs, compared with 29%
in the case of scFvs without histidine in the protein sequence
labelling protocol employing the 99mTcCO precursor for
scFvs 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-
ease of purification of the protein on a nickel affinity
column. Mixing of the scFvs together with 99mTcCO in
buffer at 37°C for 15 min results in >90% incorporation
of the total activity (Fig. 12). No protein aggregation
could 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-fold
excess of free histidine resulted in only minor dissocia-
tion of activity from the labelled scFvs, which proved
the 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 initial
activity). If the protein had endogenous histidines, less
than 25% of initial activity was incorporated. These da-
ta show that labelling occurs predominantly through the
His-tag. Further experiments showed that three histi-
dines in a row are sufficient for stable incorporation of
99mTcCO. The in vitro immunoreactivity of a series of
seven different labelled scFvs ranged from 57% to 97%
(Table 3). The in vivo stability of labelled scFvs was
tested with an anti erbB2/Her2 scFv 4D5 in tumour-
bearing nu/nu mice. Analysis of the whole-mouse sera
after 1 h revealed that 75% of the injected activity was
still migrating with the intact scFvs, whereas the rest of
the activity was bound to albumin and high-molecular-
weight proteins. The tumour localisation was 1.4%

European Journal of Nuclear Medicine Vol. 29, No. 11, November 2002

i.d./g 24 h post injection, giving rise to tumour/blood
ratios of approximately 9. Elevated renal uptake and
prolonged retention were detected (108.6%±21.7%
i.d./g), which presumably represents an inherent clear-
ance property of scFvs. The reason why radioiodinated
scFvs 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 succinylation
of the lysine side-chain and co-administration of excess
L-lysine, can significantly reduce the high renal uptake
of technetium-tricarbonyl labelled scFvs by a factor of
approximately 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]. MUC1
mucin is up-regulated and abnormally glycosylated in
bladder cancer and is a promising target for intravesical
radioimmunotherapy. The preliminary results clearly re-
vealed better retention of immunoreactivity of the
188ReCO-labelled mAb (80% at 48 h post labelling)
compared with the 2-mercaptoethanol-reduced and
Re(V)-labelled mAb (<20% at 48 h post labelling). This
suggests that the carbonyl labelling methodology may be
more appropriate for intravesical radioimmunotherapy
using 188Re.
Certainly more efforts are required to improve the bi-
ological characteristics of Tc/ReCO-labelled antibodies
and scFvs in order to permit routine clinical and eventu-
ally therapeutic application. However, the stability and
efficiency of this gentle labelling technique for scFvs
and antibodies is a decisive advantage.
Miscellaneous examples of organometallic
radiolabelled biomolecules
Apart from organometallic technetium and rhenium
complexes, there have been only a few reports on other
organometallic compounds of radionuclides for potential
radiopharmaceutical 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 therapeutic
application, 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) for
myocardial imaging agent had to be abandoned because
of the high accumulation of the compound in the liver
and the slow clearance from the blood pool. Reversible
association of the (CH3)2[67Ga]+ core with red blood
cells was found to be the reason for the disappointing re-
sults [67].
Wenzel et al. have described the potential of 97Ru- or
103Ru-ruthenocene-glycine derivatives (“Ru-ruppuran”)
European Journal of Nuclear Medicine Vol. 29, No. 11, November 2002
1539
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 longer
half-life of the ruthenium isotopes compared with io-
dine-123 make them attractive. The renal clearance rate
of the organometallic derivatives has been compared
with that of 125I-hippuran in rabbits. A similar renal and
plasma clearance pattern was found for 103Ru-ruppuran
and 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. The
authors claim that the absorbed dose in the bladder and
the kidneys would be slightly lower for 97Ru-ruppuran
than for conventional 123I-hippuran.
Conclusion
It is apparent that organometallic compounds are a valu-
able and serious alternative to state of the art labelling
techniques. The encouraging results of preclinical and
clinical studies with organometallic labelled, tumour
affine peptides and scFv fragments form the basis for
further investigations. The potential for use of organo-
metallic labelling techniques in nuclear medicine will
also depend on the therapeutic success of organometallic
compounds and the availability of appropriate radionucl-
ides. In the future, chemists and radiopharmacists will be
equally challenged to exploit the aqueous organometallic
chemistry of potential radionuclides to develop novel
techniques 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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