In vitro and in vivo evaluation of the bifunctional chelator NODIA-Me in combination with a prostate-specific membrane antigen targeting vector
Introduction
Obtaining maximum information from positron emission tomography (PET) imaging relies on the development of target-specific radiotracers. Besides compounds labelled with the PET nuclides carbon-11 and fluorine-18, radiopharmaceuticals labelled with positron-emitting radiometals are an important part of ongoing research. In particular, the two radiometals gallium-68 and copper-64 have received increased attention in the development of new PET radiopharmaceuticals. One general advantage over carbon-11 and fluorine-18 is that labelling reactions using radiometals can typically be carried out in aqueous media and are therefore amenable to kit formulations. The interest in gallium-68 originates from its physical properties (t1/2 = 67.71 min, β+ 89%, E(β+)max = 1.9 MeV), which are suitable for use with targeting vectors possessing rapid pharmacokinetics such as radiotracers based on small molecules and peptides [[1], [2], [3], [4]]. In contrast to carbon-11 and fluorine-18, the radiometal gallium-68 is generator produced and readily accessible without the need for an on-site cyclotron. More recently, a GMP-compliant pharmaceutical grade generator produced by Eckert & Ziegler has received marketing authorization. Other clinical grade gallium-68 generators are in development, which will spur the continued use of 68Ga-based radiopharmaceuticals in the near future [5].
The cyclotron-produced radiometal copper-64 has also shown promise as both a suitable PET imaging and a therapeutic radionuclide due to its favorable decay characteristics (t1/2 = 12.7 h, β+ 17.4%, E(β+)max = 0.656 MeV; β− 39%, E(β−)max = 0.573 MeV) and commercial availability in large quantities with high specific activity [[6], [7], [8]]. The intermediate half-life of copper-64 allows visualization of processes at time points up to 48 h, which is ideal for developing radiopharmaceuticals that require longer circulation times to achieve optimal uptake. The half-life of copper-64 also allows central production and shipment. Furthermore, 64Cu-labelled radiopharmaceuticals may be used to provide pharmacokinetic profiles of therapeutic radiotracers that are labelled with copper-67 (t1/2 = 61.5 h, β− 100%, Emax = 0.121 MeV) as this isotope has promise in endoradiotherapy applications [9].
Radiolabelling of target-specific radiopharmaceuticals with a radiometal generally requires the use of a bifunctional chelator (BFC) that (1) forms kinetically stable complexes with the radiometal and (2) possesses a function for the covalent attachment of a target-specific molecule. High kinetic and metabolic stability of the metal chelate is essential to avoid accumulation of radioactivity in background tissues that may result from degradation or transmetallation in vivo. However, introduction of a metal chelate into a biomolecule is a non-innocent modification. Chelates/metal complexes often have an impact on the biological properties of the radiopharmaceutical. The use of different radiometals with the same BFC can alter binding affinities as illustrated by reported studies on various gallium and yttrium labelled somatostatin analogs such as DOTA-[Tyr3]-octreotate and DOTA-octreotide [10]. Variation of the metal chelate by using different BFCs with the same radiometal can also result in altered pharmacokinetic profiles [11,12]. Interestingly, the influence of the metal chelate is not limited to radiotracers based on small molecules or peptides. For example, the biodistribution profiles of 64Cu-labelled antibody fragments as well as full antibody conjugates can also be influenced by the nature of the metal chelate [13,14]. Evidently the important role that chemical structure has on the biological performance of a radiotracer demands continued development and optimization of new BFCs. The dependency of radiotracer performance on the chemical structure also presents an opportunity for radiochemists to tune the pharmacokinetics. Radiotracer design incorporates control over the metal complex properties such as overall charge, lipophilicity and size as indispensable handles for optimizing target-specific binding and distribution in vivo.
Numerous BFCs have been developed for both gallium-68 and copper-64 (Scheme 1), such as NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), NODAGA (1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid) [[15], [16], [17], [18]] [1,11,[19], [20], [21]], TRAP (1,4,7-triazacyclononane-1,4,7-tris[methyl(2-carboxyethyl)phosphinic acid) [[22], [23], [24]], PCTA (3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid) [[25], [26], [27]], and rigid cage-like sarcophagine type ligands such as diamsar [3,[28], [29], [30], [31], [32]]. The most prominent BFC for gallium-68 is the ligand DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid [6], while copper-specific ligands include CB-TE2A (2,2′-(1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diyl)diacetic acid) [28,[33], [34], [35], [36], [37], [38]], and bispidines [[39], [40], [41]]. For a more comprehensive overview, the reader is referred to recent reviews [6,[42], [43], [44], [45]].
The clinical success of 68Ga-labelled PSMA-HBED-CC (also known as DKFZ-PSMA-11) represents an archetypal example of a system where the nature of the metal chelate forms an integral part of the radiotracer's binding mode. The aromatic rings of the [68Ga]Ga-HBED-CC chelate form a key element for docking hydrophobic patches near the binding pocket of the target protein prostate-specific membrane antigen (PSMA) [46]. PSMA is an attractive molecular target for the diagnosis due to its upregulation in primary and metastatic prostate cancer [47,48]. The PSMA pocket is comprised of two sub-pockets with one surface-exposed highly positively charged pharmacophore region containing two Zn2+ ions, and a second buried hydrophobic non-pharmacophore sub-pocket [49,50]. While the urea motif of PSMA-HBED-CC binds to the pharmacophore region, the BFC HBED-CC interacts advantageously with the lipophilic part of the PSMA pocket [46,51]. In case of traditional macrocyclic BFCs like DOTA, which lack aromatic rings, lipophilic substituents were consequently introduced into the linker region in order to mimic the proven additional biologic interactions of the chelator HBED-CC [[52], [53], [54], [55], [56]].
We recently described a chelating platform based on the macrocycle 1,4,7-triazacyclononane (TACN) with additional five-membered azaheterocyclic arms including imidazole, methylimidazole and thiazole residues [57]. These ligands are characterized by their excellent complexation properties for radioactive, divalent copper cations. They label instantaneously with [64Cu]CuCl2 under very mild conditions in the pH range of 4–8 with high molar activities of 120–180 MBq nmol−1. Experiments showed that corresponding methylimidazole-type ligands (e.g. NODIA-Me in Scheme 1) that incorporate borderline azaheterocyclic N donors according to the HSAB (hard and soft acids and bases) principle form rigid and stable Ga3+ complexes allowing their application also with the PET-nuclide gallium-68 [58]. This study reports the in vitro and in vivo evaluation of the BFC NODIA-Me (2-(4,7-bis((1-methyl-1H-imidazol-2-yl)methyl)-1,4,7-triazonan-1-yl)acetic acid) 1 labelled with gallium-68 and copper-64 in combination with the glutamate-urea-lysine moiety for targeting PSMA. The aim of this study was to demonstrate that NODIA-Me is suitable for future radiopharmaceutical development.
Section snippets
General
Chemicals and solvents were purchased from Sigma-Aldrich and TCI Europe, and used as received. The precursor Glu-NH-CO-NH-Lys(Ahx)-(tBu)3 ester was purchased from ABX (Radeberg, Germany). Fmoc-3-(2-naphthyl)-L-alanine was obtained from IRIS Biotech (Marktredwitz, Germany). The bifunctional chelator NODIA-Me (2-(4,7-bis((1-methyl-1H-imidazol-2-yl)methyl)-1,4,7-triazonan-1-yl)acetic acid) 1 was prepared as previously described [58]. The radiotracer [68Ga]Ga-PSMA-HBED-CC was prepared as previously
Bioconjugate synthesis
Our previously described triply substituted ligands lack a functionality for the covalent conjugation of biologically active targeting vectors [57]. For this reason, we developed the BFC NODIA-Me 1, in which we replaced one methylimidazole arm with an acetic acid group (Scheme 2) [58]. This acetic acid group serves as site for the attachment of appropriate targeting vectors via peptide bond formation. The glutamate-urea-lysine (Glu-NH-CO-NH-Lys) structural motif is a well-known building block
Conclusions
In the present work, the novel chelator NODIA-Me 1 was conjugated to the PSMA targeting Glu-NH-CO-NH-Lys moiety to give the final bioconjugate 4, which was labelled with gallium-68 and copper-64 in molar activities of ~20 MBq nmol−1. The metal-free as well as the Ga and Cu complexes of 4 revealed different affinities to PSMA in competitive binding studies underscoring the influence of the metal chelate on biological properties. Evaluation of the 68Ga- and 64Cu-labelled conjugates by PET imaging
Acknowledgements
JPH thanks the Department of Nuclear Medicine, University Hospital Freiburg, the German Cancer Consortium (DKTK), and the German Cancer Research Center (DKFZ) for financial support as well as the Swiss National Science Foundation (SNSF Professorship PP00P2_163683) and the European Research Council (ERC-StG-2015, NanoSCAN – 676904). MDB thanks the Chemistry Department of the Albert-Ludwigs-University Freiburg (Germany) for technical support, in particular Christoph Warth for MS measurements,
References (74)
- et al.
(68)Ga-labeled DOTA-peptides and (68)Ga-labeled radiopharmaceuticals for positron emission tomography: current status of research, clinical applications, and future perspectives
Semin Nucl Med
(2011) Recent developments in the design of bifunctional chelators for metal-based radiopharmaceuticals used in positron emission tomography
Inorg Chim Acta
(2012)- et al.
Copper radionuclides and radiopharmaceuticals in nuclear medicine
Nucl Med Biol
(1996) - et al.
The ionic charge of copper-64 complexes conjugated to an engineered antibody affects biodistribution
Bioconjug Chem
(2015) - et al.
Imaging cancer using PET–the effect of the bifunctional chelator on the biodistribution of a (64)Cu-labeled antibody
Nucl Med Biol
(2011) - et al.
Optimization, biological evaluation and microPET imaging of copper-64-labeled bombesin agonists, [64Cu-NO2A-(X)-BBN(7-14)NH2], in a prostate tumor xenografted mouse model
Nucl Med Biol
(2010) - et al.
Be spoilt for choice with radiolabelled RGD peptides: preclinical evaluation of (6)(8)Ga-TRAP(RGD)(3)
Nucl Med Biol
(2013) - et al.
An improved synthesis and biological evaluation of a new cage-like bifunctional chelator, 4-((8-amino-3,6,10,13,16,19-hexaazabicyclo[6.6.6]icosane-1-ylamino)methyl)benzoic acid, for 64Cu radiopharmaceuticals
Nucl Med Biol
(2010) - et al.
Bifunctional gallium-68 chelators: past, present, and future
Semin Nucl Med
(2016) - et al.
Small molecule inhibitors of PSMA incorporating technetium-99m for imaging prostate cancer: effects of chelate design on pharmacokinetics
Inorg Chim Acta
(2012)
Technetium and gallium derived radiopharmaceuticals: comparing and contrasting the chemistry of two important radiometals for the molecular imaging era
Chem Rev
Feasibility and availability of (6)(8)Ga-labelled peptides
Eur J Nucl Med Mol Imaging
68Ga-PET: a powerful generator-based alternative to cyclotron-based PET radiopharmaceuticals
Contrast Media Mol Imaging
68Ga-Based radiopharmaceuticals: production and application relationship
Molecules
Copper chelation chemistry and its role in copper radiopharmaceuticals
Curr Pharm Des
The rise of metal radionuclides in medical imaging: copper-64, zirconium-89 and yttrium-86
Future Med Chem
Targeted radiotherapy of prostate cancer with a gastrin-releasing peptide receptor antagonist is effective as monotherapy and in combination with rapamycin
J Nucl Med
Novel Cu-64- and Ga-68-labeled RGD conjugates show improved pet imaging of alpha(v)beta(3) integrin expression and facile radiosynthesis
J Nucl Med
64Cu-labeled inhibitors of prostate-specific membrane antigen for PET imaging of prostate cancer
J Med Chem
18F, 64Cu, and 68Ga labeled RGD-bombesin heterodimeric peptides for PET imaging of breast cancer
Bioconjug Chem
68Ga-labeled multimeric RGD peptides for microPET imaging of integrin αvβ3 expression
Eur J Nucl Med Mol Imaging
Convenient preparation of 68Ga-based PET-radiopharmaceuticals at room temperature
Bioconjug Chem
Preparation of a promising angiogenesis PET imaging agent: 68Ga-labeled c(RGDyK)–isothiocyanatobenzyl-1,4,7-triazacyclononane-1,4,7-triacetic acid and feasibility studies in mice
J Nucl Med
Evaluation of 64Cu-labeled bifunctional chelate-bombesin conjugates
Bioconjug Chem
Positron emission tomography (PET) imaging of prostate cancer with a gastrin releasing peptide receptor antagonist–from mice to men
Theranostics
A triazacyclononane-based bifunctional phosphinate ligand for the preparation of multimeric 68Ga tracers for positron emission tomography
Chem A Eur J
TRAP, a powerful and versatile framework for gallium-68 radiopharmaceuticals
Chem A Eur J
Evaluation of bifunctional chelates for the development of gallium-based radiopharmaceuticals
Bioconjug Chem
(S)-5-(p-Nitrobenzyl)-PCTA, a promising bifunctional ligand with advantageous metal ion complexation kinetics
Bioconjug Chem
68Ga Small peptide imaging: comparison of NOTA and PCTA
Bioconjug Chem
Structural biology of copper trafficking
Chem Rev
Synthesis of a novel bifunctional chelator AmBaSar based on sarcophagine for peptide conjugation and (64)Cu radiolabelling
Dalton Trans
New 64Cu PET imaging agents for personalised medicine and drug development using the hexa-aza cage, SarAr
Org Biomol Chem
Synthesis of a new cage ligand, SarAr, and its complexation with selected transition metal ions for potential use in radioimaging
J Chem Soc Dalton Trans
Comparative in vivo stability of copper-64-labeled cross-bridged and conventional tetraazamacrocyclic complexes
J Med Chem
In vivo evaluation and small-animal PET/CT of a prostate cancer mouse model using 64Cu bombesin analogs: side-by-side comparison of the CB-TE2A and DOTA chelation systems
J Nucl Med
Evaluation of [64Cu]Cu-DOTA and [64Cu]Cu-CB-TE2A chelates for targeted positron emission tomography with an alphavbeta6-specific peptide
Mol Imaging
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