Efficient bifunctional gallium-68 chelators for positron emission tomography: Tris(hydroxypyridinone) ligands

Article abstract of DOI:10.1039/c1cc12123e

A new tripodal tris(hydroxypyridinone) bifunctional chelator for gallium allows easy production of ⁶⁸Ga-labelled proteins rapidly under mild conditions in high yields at exceptionally high specific activity and low concentration.

Full text of DOI:10.1039/c1cc12123e

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Efficient bifunctional gallium-68 chelators for positron emission
tomography: tris(hydroxypyridinone) ligandsw
a
a
a
David J. Berry,a Yongmin Ma,ab James R. Ballinger, Richard Tavare, Alexander Koers,
Kavitha Sunassee,a Tao Zhou,b Saima Nawaz,a Gregory E. D. Mullen,a Robert C. Hiderb
and Philip J. Blower*a
´
Received 13th April 2011, Accepted 9th May 2011
DOI: 10.1039/c1cc12123e
A new tripodal tris(hydroxypyridinone) bifunctional chelator for
gallium allows easy production of 68Ga-labelled proteins rapidly
under mild conditions in high yields at exceptionally high specific
activity and low concentration.
pH 3–5.5 and the complex has excellent stability in plasma.5
Its derivative NODAGA (3) has been conjugated to proteins
and radiolabelled with 68Ga at pH 3.5-4 in 7 min.6 Another
promising chelator for 68Ga is HBED (4), which forms a Ga
complex with a very high logK of 38.5.7 The derivative
HBED-CC TFP (5) has been used for protein labeling in
80% yield by incubating at pH 4.1 for 5 min.8 The acyclic
ligand DTPA complexes gallium with high affinity but poor
kinetic stability.9 Derivatising the carbon backbone of DTPA
can increase the stability of the 68Ga complexes. Nevertheless
these 68Ga complexes undergo significant dissociation in
serum.10 Recently the acyclic H2DEDPA (6) and a bifunctional
The germanium-68/gallium-68 generator promises to make
molecular imaging with positron emission tomography (PET)
more widely available clinically, much as the molybdenum-99/
technetium-99m generator did in the last century for single
photon imaging, by providing a convenient, economic and
reliable source of positron-emitting radionuclide without the
need for a local cyclotron.1 Gallium-68 has a half life of 68 min
and a high positron yield (90%, 1.9 MeV).2 To achieve its full
potential, incorporation of this isotope into biomolecules for
molecular imaging requires bifunctional chelators able to bind
the Ga3+ ion rapidly under mild aqueous conditions, to give a
complex with sufficient kinetic stability in vivo to withstand
challenge from plasma transferrin, allowing imaging over
several hours.
The current ‘‘gold standard’’ bifunctional 68Ga chelator is
the aminocarboxylate macrocycle DOTA (1, Fig. 1) and
67Ga-DOTA bioconjugates have been reported to be stable
in serum in vitro for at least 1250 h.3 The most well known
68Ga-DOTA bioconjugate is 68Ga-DOTATOC, a somatostatin
analogue used clinically for somatostatin receptor imaging.4
Despite the high kinetic stability of the 68Ga-DOTA complex,
DOTA has several drawbacks as a 68Ga chelator. Typical
radiolabelling conditions entail heating (e.g. 95 1C) for up to
30 min at pH 4.6. The long radiolabelling time allows extensive
decay during labelling and the high temperature and low pH
are unsuitable for many proteins of interest for molecular
imaging. Other chelators are being evaluated to circumvent
some of these problems. The macrocycle NOTA (2) can be
radiolabelled with 68Ga at room temperature within 10 min at
a King’s College London, Division of Imaging Sciences and Biomedical
Engineering, The Rayne Institute, St Thomas’ Hospital, London,
SE1 7EH, UK. E-mail: Philip.Blower@kcl.ac.uk
b King’s College London, Institute of Pharmaceutical Science,
Franklin Wilkins Building, London, SE1 9NH, UK
w Electronic supplementary information (ESI) available: Details of
chemical syntheses, radiolabelling and in vitro and in vivo studies. See
DOI: 10.1039/c1cc12123e
Fig. 1 Structures of some 68Ga chelators.
c
7068 Chem. Commun., 2011, 47, 7068–7070
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derivative have shown promise as 67/68Ga chelators, radio-
labelling at pH 4.5 in 10 min.11
The similarity in coordination chemistry between Fe3+ and
Ga3+ suggests the use of siderophore-like chelators for
gallium. Indeed the siderophore Desferrioxamine-B (DFO-B, 7)
12
was used previously as a bifunctional chelator for 67/68Ga
but blood clearance of 68Ga-DFO-Octreotide in patients is
slow.13 Deferiprone (8) is a bidentate 3-hydroxy-4-pyridinone
(HPO) that is used clinically for the treatment of iron over-
load. Deferiprone and its analogues are effective scavengers of
67Ga with the ability to remove 67Ga bound to transferrin.14
Hexadentate tris-HPO derivatives have been developed for
Fe3+ and Al3+ sequestration15 and have recently been shown
to bind gallium with high affinity.16 However, these ligands
cannot be derivatised for bioconjugation, and they are
conformationally ill-suited to the formation of mononuclear
six-coordinate complexes. Here we evaluate the use of CP256 (9),
a powerful hexadentate HPO Fe3+ chelator which overcomes
both these limitations,17 for radiolabelling with gallium
radioisotopes. We report its radiolabelling properties with
68Ga (r 5 min at room temperature and at mild pH, B6.5)
in comparison with those of other chelators widely used for
this purpose, and the stability of its 67Ga complex in biological
media (see ESIw for further information). We also report the
synthesis of its bifunctional maleimide derivative YM-103 (10)
and its use to label a thiol-containing protein C2Ac with 68Ga
as a potential PET imaging agent for cell death.
Fig.
2
Radiolabelling yield versus ligand concentration for
68Ga-DOTA (pH 4.4, 30 min, 100 1C), 68Ga-NOTA (pH 3.6, 10 min,
room temp), 68Ga-HBED (pH 4.6, 10 min, room temp) and 68Ga-CP256,
(pH 6.5, 5 min, room temp). All experiments were conducted with the
same batch of 68Ga eluate. All radiolabelling buffers were 0.2 M acetic
acid/sodium acetate.
CP256 was found to complex natural-abundance gallium
rapidly at neutral pH and room temperature. Mass spectro-
metry showed the presence of only a 1 : 1 complex at
m/z 740.3631 for singly protonated Ga-CP256 and 370.6865
for the doubly protonated species. CP256 is designed to form
neutral, six-co-ordinate complexes with 3+ metals such as
Fe3+ and Ga3+ and these results indicate such a species. No
multinuclear complexes were detected, and only a single
species was detected by HPLC. Radio-HPLC analysis after
1 min incubation of CP256 with 67Ga-citrate showed a single
radioactive peak with a retention time matching that of the
cold Ga-complex. A side-by-side comparison was made of
the 68Ga-chelating efficiency of CP256 with that of other
established Ga chelators (DOTA, NOTA and HBED), using
instant thin layer chromatography (ITLC) at progressively
lower chelator concentration, using the same 68Ga generator
(IGG100, Eckert & Ziegler, Germany) eluate for each ligand
(Fig. 2). A radiochemical yield (RCY) of 68Ga-CP256 of
98–100% was achieved at a ligand concentration of 10 mM
after 5 min, pH 6.5 at room temperature. At 1 mM, the RCY
was 73%. In contrast, the RCY of 68Ga-NOTA (at room
temp) and 68Ga-DOTA (with heating at 100 1C for 30 min at
pH 4.4) fell to approximately 80% and 95% respectively at
10 mM and 4% and 7% at 1 mM (Fig. 2). The RCY of
68Ga-HBED dropped from 96–100% at 10 mM and above to
36% at 1 mM. Further data supporting the comparative
labelling efficiency of CP256 are provided in the ESI.w
Fig.
3
Size exclusion elution profiles of (A) 67Ga-citrate and
(B) 67Ga-CP256 in buffer and apo-transferrin after 4 h incubation.
apotransferrin (a 260 fold excess in terms of Ga-binding
capacity since there are two metal binding sites per transferrin
molecule) in the presence of bicarbonate at 37 1C. Again no
transchelation was observed (Fig. 3), whereas significant
transchelation by transferrin was observed with 67Ga-citrate
as the control.
The outstanding radiolabelling kinetics of 68Ga-CP256 and
in vitro stability observed for 67Ga-CP256 encouraged us to
synthesise a bifunctional derivative for protein labelling. The
maleimide derivative 10 was chosen to confer site-specificity
on conjugation to proteins containing an engineered free
cysteine residue. To evaluate the potential of 10 for 68Ga
labelling of biomolecules, the protein C2Ac was selected.
C2Ac is an analogue of C2A (the phosphatidylserine (PS)-
binding domain of synaptotagmin I, an amphipathic protein
Incubation of 67Ga-CP256 in human serum for 4 h at 37 1C
showed no evidence of protein binding or release of 67Ga when
analysed by size exclusion chromatography. To provide a
more stringent test of resistance to transchelation with transferrin,
the complex was incubated with
a 130-fold excess of
c
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This work was conducted within the King’s College
London-UCL Comprehensive Cancer Imaging Centre supported
by Cancer Research UK & EPSRC, in association with MRC
and DoH (UK). The work was supported by an MRC
studentship to DJB and by the Centre of Excellence in Medical
Engineering funded by the Wellcome Trust and EPSRC under
grants (WT 088641/Z/09/Z). JRB was supported in part by a
National Institute for Health Research (NIHR) comprehensive
Biomedical Research Centre award to Guy’s & St Thomas’ NHS
Foundation Trust in partnership with King’s College London
and King’s College Hospital NHS Foundation Trust. YM was
supported in part by a grant from the British Heart Foundation
Centre of Excellence at King’s College London. We thank
Wellcome Trust for grant support enabling PET-CT imaging.
Selected samples were sent for mass spectral analysis at the
EPSRC Mass Spectrometry Service Centre (Swansea) and at
the Mass Spectrometry Facility, King’s College, London
(Franklin Wilkins Building).
Notes and references
z Animal studies were carried out in accordance with UK Research
Councils’ and Medical Research Charities’ guidelines on Responsibility
in the Use of Animals in Bioscience Research, under a UK Home
Office licence and were approved by the King’s College London Local
Ethics Committee.
Fig. 4 PET-CT images of mice 90 min after intravenous injection of
(A) 68Ga-YM-103-C2Ac and (B) unchelated 68Ga.
which binds to PS in a Ca2+-dependent manner and is being
investigated as an imaging agent for cell death) into which a
cysteine residue has been engineered18 for purposes of bio-
conjugate synthesis. Incubation of 10 (or its HCl addition
product, which is also formed as an intermediate during the
synthesis of 10, see ESIw) with C2Ac produced the bio-
conjugate YM-103-C2Ac whose electrospray mass spectrum
showed the incorporation of a single molecule of 10. The
conjugate (20 mg, 1 mg mlꢀ1, 63 mM) was incubated with
freshly-eluted 68Ga in 0.5 M ammonium acetate buffer,
pH B5.5, giving quantitative labelling after 5 min as deter-
mined by TLC.
1 W. A. P. Breeman and A. M. Verbruggen, Eur. J. Nucl. Med.
Mol. Imaging, 2007, 34, 97.
2 D. Reichert, J. Lewis and C. Anderson, Coord. Chem. Rev., 1999,
184, 3.
3 A. Heppeler, S. Froidevaux, H. Macke, E. Jermann, M. Behe,
¨
P. Powell and M. Hennig, Chem.–Eur. J., 1999, 5, 1974.
4 J. Kowalski, M. Henze, J. Schuhmacher, H. R. Macke,
¨
M. Hofmann and U. Haberkorn, Mol. Imaging Biol., 2003, 5, 42.
5 I. Velikyan, H. Maecke and B. Langstrom, Bioconjugate Chem.,
2008, 19, 569.
6 C. Wangler, B. Wangler, S. Lehner, A. Elsner, A. Todica,
P. Bartenstein, M. Hacker and R. Schirrmacher, J. Nucl. Med.,
2011, 52, 586.
7 Y. Sun, C. Anderson, T. Pajeau, D. Reichert, R. Hancock,
R. Motekaitis, A. Martell and M. Welch, J. Med. Chem., 1996,
39, 458.
The radiolabelled conjugate showed retention of calcium-
dependent binding to PS in a red blood cell binding assay
in vitro. A PET imaging study was therefore carried out in a
normal mouse (Fig. 4A).z After 90 min post-injection the 68Ga
was located almost exclusively in the kidney, with some
excretion to the bladder, in contrast to the distribution
throughout the whole mouse observed (Fig. 4B) when
uncomplexed 68Ga was injected. This demonstrates that
68Ga is not released from the conjugate in vivo during the
imaging period. We conclude that the tris(hydroxypyridinone)
ligand CP256 is an excellent chelator for gallium and can be
radiolabelled with gallium isotopes more quickly, with higher
yield, under milder conditions and to significantly higher
specific activity than the currently established chelators
DOTA, NOTA and HBED. The complex is extremely stable
in serum and when challenged with excess apotransferrin. The
maleimide derivative 10 can be conjugated site-specifically to
cysteine residues and the resulting bioconjugate is efficiently
labelled with 68Ga to high specific activity. It is likely to
become the bifunctional chelator of choice for labelling of
sensitive proteins with 68Ga.
8 M. Eder, B. Wangler, S. Knackmuss, F. LeGall, M. Little,
U. Haberkorn, W. Mier and M. Eisenhut, Eur. J. Nucl. Med.
Mol. Imaging, 2008, 35, 1878.
9 S. Wagner and M. Welch, J. Nucl. Med., 1979, 20, 428.
10 B. Koop, S. N. Reske and B. Neumaier, Radiochim. Acta, 2007, 95,
39.
11 E. Boros, C. L. Ferreira, J. F. Cawthray, E. W. Price,
B. O. Patrick, D. W. Wester, M. J. Adam and C. Orvig, J. Am.
Chem. Soc., 2010, 132, 15726.
12 P. Smith-Jones, B. Stolz, C. Bruns, R. Albert, H. Reist, R. Fridrich
and H. Macke, J. Nucl. Med., 1994, 35, 317.
¨
13 K. Eisenwiener, M. Prata, I. Buschmann, H. Zhang, A. Santos,
S. Wenger, J. Reubi and H. Macke, Bioconjugate Chem., 2002, 13,
530.
¨
14 M. Santos, M. Gil, S. Marques, L. Gano, G. Cantinho and
S. Chaves, J. Inorg. Biochem., 2002, 92, 43.
15 S. Chaves, S. M. Marques, A. M. F. Matos, A. Nunes, L. Gano,
T. Tuccinardi, A. Martinelli and M. A. Santos, Chem.–Eur. J.,
2010, 16, 10535.
16 S. Chaves, A. C. Mendonc¸ a, S. M. Marques, M. I. Prata,
A. C. Santos, A. F. Martins, C. F. G. C. Geraldes and
M. A. Santos, J. Inorg. Biochem., 2011, 105, 31.
17 T. Zhou, H. Neubert, D. Y. Liu, Z. D. Liu, Y. M. Ma, X. L. Kong,
W. Luo, S. Mark and R. C. Hider, J. Med. Chem., 2006, 49, 4171.
18 R. Tavare, R. Torres Martin De Rosales, P. J. Blower and G. E.
´
D. Mullen, Bioconjugate Chem., 2009, 20, 2071.
c
7070 Chem. Commun., 2011, 47, 7068–7070
This journal is The Royal Society of Chemistry 2011