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

Abstract

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.

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 °C) 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 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 derivative have shown promise as ^67/68Ga chelators, radio-labelling at pH 4.5 in 10 min.¹¹

Fig. 1. Structures of some ⁶⁸Ga chelators.

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) was used previously as a bifunctional chelator for ^67/68Ga ¹² 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 overload. 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 (≤ 5 min at room temperature and at mild pH, ~6.5) in comparison with those of other chelators widely used for this purpose, and the stability of its ⁶⁷Ga complex in biological media (see ESI 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.

CP256 was found to complex natural-abundance gallium rapidly at neutral pH and room temperature. Mass spectrometry 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 μM after 5 min, pH 6.5 at room temperature. At 1 μM, the RCY was 73%. In contrast, the RCY of ⁶⁸Ga-NOTA (at room temp) and ⁶⁸Ga-DOTA (with heating at 100 °C for 30 min at pH 4.4) fell to approximately 80% and 95% respectively at 10 μM and 4% and 7% at 1 μM (Fig. 2). The RCY of ⁶⁸Ga-HBED dropped from 96–100% at 10 μM and above to 36% at 1 μM. Further data supporting the comparative labelling efficiency of CP256 are provided in the ESI.

Fig. 2. Radiolabelling yield versus ligand concentration for ⁶⁸Ga-DOTA (pH 4.4, 30 min, 100 °C), 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.

Incubation of ⁶⁷Ga-CP256 in human serum for 4 h at 37 °C 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 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 °C. Again no transchelation was observed (Fig. 3), whereas significant transchelation by transferrin was observed with ⁶⁷Ga-citrate as the control.

Fig. 3. Size exclusion elution profiles of (A) ⁶⁷Ga-citrate and (B) ⁶⁷Ga-CP256 in buffer and apo-transferrin after 4 h incubation.

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 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 bioconjugate synthesis. Incubation of 10 (or its HCl addition product, which is also formed as an intermediate during the synthesis of 10, see ESI) with C2Ac produced the bioconjugate YM-103-C2Ac whose electrospray mass spectrum showed the incorporation of a single molecule of 10. The conjugate (20 μg, 1 mg ml⁻¹, 63 μM) was incubated with freshly-eluted ⁶⁸Ga in 0.5 M ammonium acetate buffer, pH ~5.5, giving quantitative labelling after 5 min as determined by TLC.

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).^‡ 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.

Fig. 4. PET-CT images of mice 90 min after intravenous injection of (A) ⁶⁸Ga-YM-103-C2Ac and (B) unchelated ⁶⁸Ga.