TRAP, a powerful and versatile framework for gallium-68 radiopharmaceuticals

Article abstract of DOI:10.1002/chem.201103503

A veritable gallium TRAP: Triazacyclononane-phosphinic acid chelators (TRAP) form highly stable complexes with Ga³⁺ (see figure) extremely efficiently over a wide pH range. Homo- and heteromultimeric bioconjugates can be synthesized in a straig

Full text of DOI:10.1002/chem.201103503

DOI: 10.1002/chem.201103503
TRAP, a Powerful and Versatile Framework for Gallium-68
Radiopharmaceuticals**
[a, b]
Johannes Notni,*[a] Jakub Simecˇek,
Petr Hermann,[b] and Hans-Jꢀrgen Wester[a]
ˇ
Dedicated to Prof. em. Dr. rer. nat. habil. Ernst Anders on the occasion of his 70th birthday
The remarkable success of 68Ga-labeled peptides in neuro-
endocrine tumor diagnostics gave a major boost to the de-
velopment of 68Ga-labeled tracers for positron emission to-
mography (PET).[1a] In contrast to common PET isotopes,
68
1
Ga (t = =68 min; Eb+,max =1.89 MeV) is not produced by
2
on-site cyclotrons but, similarly to 99mTc production for scin-
tigraphy, is obtained from 68Ge/68Ga radioisotope generators
(68Ge)=270.8 days). Conjugation of biologically active
1
(t
2
molecules to bifunctional chelate ligands and subsequent
68Ga3+ complexation delivers radiopharmaceuticals that
bind to pathologically overexpressed target structures with
high affinity and thus allow for non-invasive molecular
imaging in patients.
Until now, the design of 68Ga tracers mainly relied on
DOTA[1] (Scheme 1), which is ideally suited for larger metal
ions (particularly lanthanides) but less so for the smaller
Ga3+ ion. However, the utility of the smaller NOTA[2a]
system (Scheme 1) for Ga3+ complexation has been shown
two decades ago.[2] As it has been found that it forms GaIII
complexes with higher kinetic stability and allows higher
68Ga labeling yields, conjugates of some of its bifunctional
derivatives (mainly NODAGA,[2b] Scheme 1) are increasing-
ly utilized in the development of 68Ga radiopharmaceuticals
in recent times. Inspired by these results, we report here the
synthesis, properties, and preclinical evaluation of 68Ga radi-
opharmaceuticals derived from NOTA-analogous Tri-Azacy-
clononane-Phosphinic acids[3] (abbreviated TRAP, see
Scheme 1 and Table 1) and reveals their enormous utility for
nuclear medicine and molecular imaging.
Scheme 1. Macrocyclic chelators discussed in the text (see also Table 1).
Table 1. TRAP ligands used in this study.
Identifier
Substituent R
(Scheme 1)
Ref.
AHCTUNGTRENNUNG
[3a]
[3b,c]
[4]
TRAP-H
TRAP-Ph
TRAP-OH
TRAP-Pr
H
Ph
CH2–OH
CH2–CH2–COOH
[5]
The 68Ga3+ complexation behavior was compared for
TRAP ligands (Table 1), DOTA, and NOTA. Figure 1
shows that TRAP ligands are able to incorporate 68Ga
nearly quantitatively (>95%) at much lower ligand concen-
trations (cL) and, due to the low pKa of phosphinic acid moi-
eties (ca. 0.7–1.5[4,5]), also in strongly acidic media; in the
case of TRAP-OH[4] and TRAP-Pr[5] even at a pH as low as
0.5. As this allows 68Ga labeling by directly using the neat
eluate (0.1m HCl) of the very popular Obninsk generator,
greatly facilitated kit production of 68Ga tracers could be
achieved. Figure 2 illustrates that particularly TRAP-Pr rap-
idly incorporates 68Ga at 258C and cL =3 mm, whereas nei-
ther DOTA nor NOTA can be labeled under these condi-
tions. However, in line with previous reports[2c] we found
that unlike DOTA, NOTA can be readily labeled at room
temperature when using a fairly high ligand concentration
of cL =100 mm (see the Supporting Information for details).
ˇ
ˇ
[a] Dr. J. Notni, J. Simecek, Prof. Dr. H.-J. Wester
Lehrstuhl fꢀr Pharmazeutische Radiochemie
Technische Universitꢁt Mꢀnchen
Walther-Meissner-Strasse 3, 85748 Garching (Germany)
and
Klinik fꢀr Nuklearmedizin
Technische Universitꢁt Mꢀnchen
Ismaninger Strasse 22, 81675 Mꢀnchen (Germany)
E-mail: johannes.notni@tum.de
ˇ
ˇ
[b] J. Simecek, Prof. Dr. P. Hermann
Department of Inorganic Chemistry, Faculty of Science
Charles University in Prague (Czech Republic)
[**] TRAP=Triazacyclononane-phosphinic acid.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201103503.
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COMMUNICATION
Figure 1. 68Ga incorporation (%) by DOTA, NOTA, and TRAP ligands
(20–30 MBq =^ 0.2–0.3 pmol of 68Ga at 958C, 5 min) as functions of cL
(top, pH 3.3) and pH (bottom, cL =10 mm =^ nL =1 nmol).
Figure 2. 68Ga incorporation (%) by DOTA, NOTA, and TRAP ligands
as functions of time (20–30 MBq =^ 0.2–0.3 pmol of 68Ga at 258C, pH 3.3),
top: cL =3 mm =^ nL =300 pmol, bottom: cL =100 mm =^ nL =10 nmol.
In addition to its remarkable labeling properties, TRAP-
Pr is particularly suitable for innovative tracer design since
it can be derivatized easily by amide formation without af-
fecting the integrity of the complexation site.[5] Integrins,
heterodimeric transmembrane adhesion receptors, are cur-
rently of particular interest for molecular imaging since they
are involved in fundamental biological processes such as cell
adhesion, migration, and proliferation. Cyclic pentapeptides
and have been widely employed in clinical studies address-
ing avb3 integrin status in angiogenic, oncological, and car-
diological evaluations.[6] Furthermore, multiple conjugates
(multimers) of RGD peptides exhibited enhanced affinities
due to the avidity effect, and thus appear better suited for in
vivo imaging of integrin expression.[7]
TRAP
ACHTUNGRTENUN(NG RGD)3 (Scheme 2) combines multimericity with
of the cycloACHTUNGTRENNUNG(RGDfK) type possess a high integrin affinity
the exceptional 68Ga3+ complexation performance of TRAP
Scheme 2. TRAPACHTUNGTRENNUNG(RGD)3, a TRAP-based cycloACHTUNGTNER(NUGN RGDfK) trimer.
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J. Notni et al.
ligands. According to current GMP standards for clinical
routine production, 68Ga labeling of TRAP
(RGD)3 was
done on a fully automated system (see the Supporting Infor-
mation). Without the necessity of pre-purification or con-
centration of the generator eluate prior to labeling, 68Ga-
higher tumor uptake of 68Ga-TRAP
ACTHNGUTERN(NUG RGD)3 (Table 3). PET
imaging (Figure 3) shows a markedly increased uptake of
the trimer in other organs such as thyroid or intestine; how-
G
Table 3. Octanol–water partition coefficients, IC50, tumor uptake (% in-
jected dose per g tumor), uptake ratios tumor/blood and tumor/muscle
90 min after injection of RGD radiotracers. IC50 values were determined
by using non-active reference compounds (natGa/19F-labeled) in competi-
tion assays on M21 cells against 125I-Echistatin.
TRAPACHTUNGTRENNUNG(RGD)3 was produced in high yields and with ex-
tremely high specific activity (SA) exceeding 4 TBqmmolÀ1
(Table 2), thus enabling production of high specific activity
Tracer
logPow
IC50 ID/g [%]
[nm] (M21)
M21/
blood
M21/
muscle
Table 2. Radiochemical yields for automated 68Ga-TRAP-(RGD)3 pro-
duction and specific activities at injection time (i.e., decay corrected to
30 min after start of synthesis). Synthesis on GallElut+ module using a
SnO2-based 68Ge/68Ga generator (iThembaLABS) eluted with 1.0m HCl;
eluate fraction of 1.25 mL (ca. 1 GBq ~10 pmol of 68Ga) with 270 mg
HEPES and 220 mL water, pH 2, reaction at 95–1008C for 5 min (see the
Supporting Information for details); radiochemical purity was always
>99.0% by radio-TLC and >99.8% by radio-HPLC.
G
68Ga-TRAP
ACHTUNGTRENNUNG
18F-Galacto-RGD
À3.2[9b]
319 1.35Æ0.53 6.1Æ3.3
7.6Æ1.7
[a] IC50 of the Ga-free compound is 43 nm.
ever, this uptake was proven to be highly specific, as virtual-
ly complete blocking was observed upon co-injection of
5 mg of unlabeled precursor per kg body weight.
After functionalization of TRAP-Pr with propargylamine,
the synthetic performance of click chemistry (CuAAC) can
be utilized for the bioconjugation to the TRAP ligand,
which has been illustrated by the synthesis of an RGD-
’click’-trimer (Scheme 3, see the Supporting Information for
details). Further examples for the versatility of the TRAP
ligand are a TRAP-biotin trimer to be used in avidin-biotin
TRAP
[nmol]
(RGD)3
Radiochemical yield
Specific activity
G
[%]
MBq
GBqmmolÀ1
1.00
0.33
0.10
95.2Æ1.7
90.0Æ2.7
65.8Æ5.6
701Æ12
663Æ20
485Æ41
701Æ12
2009Æ61
4848Æ414
68Ga tracers also using low 68Ga activities for synthesis. As
higher specific activity is tantamount to smaller absolute
amounts of substance administered, TRAP radiopharma-
ceuticals are now coming much closer to the ꢃideal tracerꢄ
concept: allowing the monitoring of biochemistry in vivo
without disturbing the underly-
targeting systems and a TRAPACHTNUGRTENUNG(RGD)2(rhodamine) hetero-
multimer, which emphasizes the facile accessibility of
ing biological processes and
equilibria. Furthermore, the
probability of undesired phar-
macological effects occuring in
human application is greatly re-
duced. Another advantage for
modern preclinical small-animal
imaging is the virtual elimina-
tion of disturbing saturation ef-
fects, which are frequently oc-
curing and caused by too low
specific activity,[8] such as com-
petition of the unlabeled com-
pound on receptors.
Comparison of 68Ga-TRAP-
ACHTUNGTRENNUNG(RGD)3 with the clinically
tested 18F-Galacto-RGD[9] (mo-
nomer; structure given in the
Supporting Information) in bio-
distribution studies using athy-
mic nude mice bearing M21
(avb3-positive) and M21L (very
low avb3-expressing) human
melanoma xenografts on right
and left shoulders and competi-
tion assays on M21 cells re-
vealed a 7.3-fold higher avb3-in-
Scheme 3. Synthesis of a TRAP-based RGD trimer employing CuII-catalyzed alkyne–azide cycloaddition
tegrin affinity and 3.9-fold (CuAAC).
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Macrocycles for 68Ga Radiopharmaceuticals
COMMUNICATION
TRAP-based tracers for the increasingly important field of
multimodal imaging (Scheme 4). These conjugates incorpo-
rated 68Ga with similar efficiency as TRAP
ACTHNUTRGNEN(UG RGD)3.
We conclude that TRAP ligands are exceptionally well
suited for the development of 68Ga tracers for in vivo molec-
ular imaging with PET, as they allow highly efficient 68Ga3+
complexation in a wide pH range (0.5–5) and at 10–30-fold
lower concentrations than NOTA and DOTA, the currently
most frequently used 68Ga chelator systems. For the example
of the TRAP-based cyclo
(RGD)3 it was shown that multimeric TRAP tracers are
(RGDfK) trimer 68Ga-TRAP-
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
easily accessible and can be 68Ga-labeled in full automation,
thereby reaching hitherto unparalleled specific activities of
several TBqmmolÀ1 without separation from excess precur-
sor. Utility of the resulting radiotracer was proven in a pre-
clinical imaging study. Further examples demonstrated the
versatility of the TRAP system in tailoring molecular imag-
ing agents and multimodal probes, all of which is justifying
high expectations for future applications of TRAP.
Figure 3. microPET scans (MIP, 75 min p. i.) of nude mouse bearing
tumor xenografts on both shoulders (left: M21L, right: M21).
Acknowledgements
Financial support by GACR (No. 203/09/1056), MSMT CR (No.
MSM0021620857), GAUK (No. 19310), EU COST Actions D38 and
PhoSciNet, and by the German Research Foundation (DFG SFB 824/
Z1) is gratefully acknowledged. We thank K. Pohle for determining the
integrin affinities, and Dr. J. Plutnar and O. Zemek, Prague, for acquisi-
tion of the NMR spectra.
Keywords: gallium-68 · macrocyclic ligands · molecular
imaging
·
positron
emission
tomography
·
radiopharmaceuticals
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Received: November 7, 2011
Published online: December 6, 2011
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Chem. Eur. J. 2011, 17, 14718 – 14722