Tetraamine-derived bifunctional chelators for technetium-99m labelling: synthesis, bioconjugation and evaluation as targeted SPECT imaging probes for GRP-receptor-positive tumours

Owing to its optimal nuclear properties, ready availability, low cost and favourable dosimetry, (99m)Tc continues to be the ideal radioisotope for medical-imaging applications. Bifunctional chelators based on a tetraamine framework exhibit facile complexation with Tc(V)O(2) to form monocationic species with high in vivo stability and significant hydrophilicity, which leads to favourable pharmacokinetics. The synthesis of a series of 1,4,8,11-tetraazaundecane derivatives (01-06) containing different functional groups at the 6-position for the conjugation of biomolecules and subsequent labelling with (99m)Tc is described herein. The chelator 01 was used as a starting material for the facile synthesis of chelators functionalised with OH (02), N(3) (04) and O-succinyl ester (05) groups. A straightforward and easy synthesis of carboxyl-functionalised tetraamine-based chelator 06 was achieved by using inexpensive and commercially available starting materials. Conjugation of 06 to a potent bombesin-antagonist peptide and subsequent labelling with (99m)Tc afforded the radiotracer (99m)Tc-N4-BB-ANT, with radiolabelling yields of >97% at a specific activity of 37 GBq micromol(-1). An IC(50) value of (3.7+/-1.3) nM was obtained, which confirmed the high affinity of the conjugate to the gastrin-releasing-peptide receptor (GRPr). Immunofluorescence and calcium mobilisation assays confirmed the strong antagonist properties of the conjugate. In vivo pharmacokinetic studies of (99m)Tc-N4-BB-ANT showed high and specific uptake in PC3 xenografts and in other GRPr-positive organs. The tumour uptake was (22.5+/-2.6)% injected activity per gram (% IA g(-1)) at 1 h post injection (p.i.). and increased to (29.9+/-4.0)% IA g(-1) at 4 h p.i. The SPECT/computed tomography (CT) images showed high tumour uptake, clear background and negligible radioactivity in the abdomen. The promising preclinical results of (99m)Tc-N4-BB-ANT warrant its potential candidature for clinical translation.

Switch from antagonist to agonist after addition of a DOTA chelator to a somatostatin analog

Peptide receptor targeting has become an increasingly attractive method to target tumors diagnostically and radiotherapeutically. Peptides linked to a variety of chelators have been developed for this purpose. They have, however, rarely been tested for their agonistic or antagonistic properties. We report here on a somatostatin antagonist that switched to an agonist upon coupling to a DOTA chelator.

Ultrastructural changes in diaphragm neuromuscular junctions in a severe mouse model for Spinal Muscular Atrophy and their prevention by bifunctional U7 snRNA correcting SMN2 splicing

In Spinal Muscular Atrophy (SMA), the SMN1 gene is deleted or inactivated. Because of a splicing problem, the second copy gene, SMN2, generates insufficient amounts of functional SMN protein, leading to the death of spinal cord motoneurons. For a "severe" mouse SMA model (Smn -/-, hSMN2 +/+; with affected pups dying at 5-7 days), which most closely mimicks the genetic set-up in human SMA patients, we characterise SMA-related ultrastructural changes in neuromuscular junctions (NMJs) of two striated muscles with discrete functions. In the diaphragm, but not the soleus muscle of 4-days old SMA mice, mitochondria on both sides of the NMJs degenerate, and perisynaptic Schwann cells as well as endoneurial fibroblasts show striking changes in morphology. Importantly, NMJs of SMA mice in which a modified U7 snRNA corrects SMN2 splicing and delays or prevents SMA symptoms are normal. This ultrastructural study reveals novel features of NMJ alterations - in particular the involvement of perisynaptic Schwann cells - that may be relevant for human SMA pathogenesis.

Ca(2+) release from the sarcoplasmic reticulum activated by the low affinity Ca(2+) chelator TPEN in ventricular myocytes

The Ca(2+) content of the sarcoplasmic reticulum (SR) of cardiac myocytes is thought to play a role in the regulation and termination of SR Ca(2+) release through the ryanodine receptors (RyRs). Experimentally altering the amount of Ca(2+) within the SR with the membrane-permeant low affinity Ca(2+) chelator TPEN could improve our understanding of the mechanism(s) by which SR Ca(2+) content and SR Ca(2+) depletion can influence Ca(2+) release sensitivity and termination. We applied laser-scanning confocal microscopy to examine SR Ca(2+) release in freshly isolated ventricular myocytes loaded with fluo-3, while simultaneously recording membrane currents using the whole-cell patch-clamp technique. Following application of TPEN, local spontaneous Ca(2+) releases increased in frequency and developed into cell-wide Ca(2+) waves. SR Ca(2+) load after TPEN application was found to be reduced to about 60% of control. Isolated cardiac RyRs reconstituted into lipid bilayers exhibited a two-fold increase of their open probability. At the low concentration used (20-40muM), TPEN did not significantly inhibit the SR-Ca(2+)-ATPase in SR vesicles. These results indicate that TPEN, traditionally used as a low affinity Ca(2+) chelator in intracellular Ca(2+) stores, may also act directly on the RyRs inducing an increase in their open probability. This in turn results in an increased Ca(2+) leak from the SR leading to its Ca(2+) depletion. Lowering of SR Ca(2+) content may be a mechanism underlying the recently reported cardioprotective and antiarrhythmic features of TPEN.

Aeromonas exoenzyme T of Aeromonas salmonicida is a bifunctional protein that targets the host cytoskeleton

Type III protein secretion has been shown recently to be important in the virulence of the fish pathogen Aeromonas salmonicida. The ADP-ribosylating toxin Aeromonas exoenzyme T (AexT) is one effector protein targeted for secretion via this system. In this study, we identified muscular and nonmuscular actin as substrates of the ADP-ribosylating activity of AexT. Furthermore, we show that AexT also functions as a GTPase-activating protein (GAP), displaying GAP activity against monomeric GTPases of the Rho family, specifically Rho, Rac, and Cdc42. Transfection of fish cells with wild type AexT resulted in depolymerization of the actin cytoskeleton and cell rounding. Point mutations within either the GAP or the ADP-ribosylating active sites of AexT (Arg-143 as well as Glu-398 and Glu-401, respectively) abolished enzymatic activity, yet did not prevent actin filament depolymerization. However, inactivation of the two catalytic sites simultaneously did. These results suggest that both the GAP and ADP-ribosylating domains of AexT contribute to its biological activity. This is the first bacterial virulence factor to be described that has a specific actin ADP-ribosylation activity and GAP activity toward Rho, Rac, and Cdc42, both enzymatic activities contributing to actin filament depolymerization.

Macrocyclic chelator-coupled gastrin-based radiopharmaceuticals for targeting of gastrin receptor-expressing tumours

PURPOSE: Diethylenetriamine-pentaacetic acid (DTPA)-coupled minigastrins are unsuitable for therapeutic application with the available beta-emitting radiometals due to low complex stability. Low tumour-to-kidney ratio of the known radiopharmaceuticals is further limiting their potency. We used macrocyclic chelators for coupling to increase complex stability, modified the peptide sequence to enhance radiolytic stability and studied tumour-to-kidney ratio and metabolic stability using (111)In-labelled derivatives. METHODS: Gastrin derivatives with decreasing numbers of glutamic acids were synthesised using (111)In as surrogate for therapeutic radiometals for in vitro and in vivo studies. Gastrin receptor affinities of the (nat)In-metallated compounds were determined by receptor autoradiography using (125)I-CCK as radioligand. Internalisation was evaluated in AR4-2J cells. Enzymatic stability was determined by incubating the (111)In-labelled peptides in human serum. Biodistribution was performed in AR4-2J-bearing Lewis rats. RESULTS: IC(50) values of the (nat)In-metallated gastrin derivatives vary between 1.2 and 4.8 nmol/L for all methionine-containing derivatives. Replacement of methionine by norleucine, isoleucine, methionine-sulfoxide and methionine-sulfone resulted in significant decrease of receptor affinity (IC(50) between 9.9 and 1,195 nmol/L). All cholecystokinin receptor affinities were >100 nmol/L. All (111)In-labelled radiopeptides showed receptor-specific internalisation. Serum mean-life times varied between 2.0 and 72.6 h, positively correlating with the number of Glu residues. All (111)In-labelled macrocyclic chelator conjugates showed higher tumour-to-kidney ratios after 24 h (0.37-0.99) compared to (111)In-DTPA-minigastrin 0 (0.05). Tumour wash out between 4 and 24 h was low. Imaging studies confirmed receptor-specific blocking of the tumour uptake. CONCLUSIONS: Reducing the number of glutamates increased tumour-to-kidney ratio but resulted in lower metabolic stability. The properties of the macrocyclic chelator-bearing derivatives make them potentially suitable for clinical purposes.

Metal-ion-dependent biological properties of a chelator-derived somatostatin analogue for tumour targeting

Somatostatin-based radioligands have been shown to have sensitive imaging properties for neuroendocrine tumours and their metastases. The potential of [(55)Co(dotatoc)] (dotatoc =4,7,10-tricarboxymethyl-1,4,7,10-tetraazacyclododecane-1-ylacetyl-D-Phe-(Cys-Tyr-D-Trp-Lys-Thr-Cys)-threoninol (disulfide bond)) as a new radiopharmaceutical agent for PET has been evaluated. (57)Co was used as a surrogate of the positron emitter (55)Co and the pharmacokinetics of [(57)Co(dotatoc)] were investigated by using two nude mouse models. The somatostatin receptor subtype (sst1-sst5) affinity profile of [(nat)Co(dotatoc)] on membranes transfected with human somatostatin receptor subtypes was assessed by using autoradiographic methods. These studies revealed that [(57)Co(dotatoc)] is an sst2-specific radiopeptide which presents the highest affinity ever found for the sst2 receptor subtype. The rate of internalisation into the AR4-2J cell line also was the highest found for any somatostatin-based radiopeptide. Biodistribution studies, performed in nude mice bearing an AR4-2J tumour or a transfected HEK-sst2 cell-based tumour, showed high and specific uptake in the tumour and in other sst-receptor-expressing tissues, which reflects the high receptor binding affinity and the high rate of internalisation. The pharmacologic differences between [(57)Co(dotatoc)] and [(67)Ga(dotatoc)] are discussed in terms of the structural parameters found for the chelate models [Co(II)(dota)](2-) and [Ga(III)(dota)](-) whose X-ray structures have been determined. Both chelates show six-fold coordination in pseudo-octahedral arrangements.

Composition Space Analysis in the Development of Copper Molybdate Hybrids Decorated by a Bifunctional Pyrazolyl/1,2,4-Triazole Ligand

A bitopic ligand, 4-(3,5-dimethylpyrazol-4-yl)-1,2,4-triazole (Hpz-tr) (1), containing two different heterocyclic moieties was employed for the design of copper(II)–molybdate solids under hydrothermal conditions. In the multicomponent CuII/Hpz-tr/MoVI system, a diverse set of coordination hybrids, [Cu(Hpz-tr)2SO4]·3H2O (2), [Cu(Hpz-tr)Mo3O10] (3), [Cu4(OH)4(Hpz-tr)4Mo8O26]·6H2O (4), [Cu(Hpz-tr)2Mo4O13] (5), and [Mo2O6(Hpz-tr)]·H2O (6), was prepared and characterized. A systematic investigation of these systems in the form of a ternary crystallization diagram approach was utilized to show the influence of the molar ratios of starting reagents, the metal (CuII and MoVI) sources, the temperature, etc., on the reaction products outcome. Complexes 2–4 dominate throughout a wide crystallization range of the composition triangle, while the other two compounds 5 and 6 crystallize as minor phases in a narrow concentration range. In the crystal structures of 2–6, the organic ligand behaves as a short [N–N]-triazole linker between metal centers Cu···Cu in 2–4, Cu···Mo in 5, and Mo···Mo in 6, while the pyrazolyl function remains uncoordinated. This is the reason for the exceptional formation of low-dimensional coordination motifs: 1D for 2, 4, and 6 and 2D for 3 and 5. In all cases, the pyrazolyl group is involved in H bonding (H-donor/H-acceptor) and is responsible for π–π stacking, thus connecting the chain and layer structures in more complicated H-bonding architectures. These compounds possess moderate thermal stability up to 250–300 °C. The magnetic measurements were performed for 2–4, revealing in all three cases antiferromagnetic exchange interactions between neighboring CuII centers and long-range order with a net moment below Tc of 13 K for compound 4.

Development of a potent DOTA-conjugated bombesin antagonist for targeting GRPr-positive tumours

Radiolabelled somatostatin-based antagonists show a higher uptake in tumour-bearing mouse models than agonists of similar or even distinctly higher receptor affinity. Very similar results were obtained with another family of G protein-coupled receptor ligands, the bombesin family. We describe a new conjugate, RM2, with the chelator DOTA coupled to D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH(2) via the cationic spacer 4-amino-1-carboxymethyl-piperidine for labelling with radiometals such as (111)In and (68)Ga.

[(99m)Tc]Demomedin C, a radioligand based on human gastrin releasing peptide(18-27): synthesis and preclinical evaluation in gastrin releasing peptide receptor-expressing models

The synthesis and preclinical evaluation of [(99m)Tc]Demomedin C in GRPR-expressing models are reported. Demomedin C resulted by coupling a Boc-protected N(4)-chelator to neuromedin C (human GRP(18-27)), which, after (99m)Tc-labeling, afforded [(99m)Tc]Demomedin C. Demomedin C showed high affinity and selectivity for the GRPR during receptor autoradiography on human cancer samples (IC(50) in nM: GRPR, 1.4 ± 0.2; NMBR, 106 ± 18; and BB(3)R, >1000). It triggered GRPR internalization in HEK-GRPR cells and Ca(2+) release in PC-3 cells (EC(50) = 1.3 nM). [(99m)Tc]Demomedin C rapidly and specifically internalized at 37 °C in PC-3 cells and was stable in mouse plasma. [(99m)Tc]Demomedin C efficiently and specifically localized in human PC-3 implants in mice (9.84 ± 0.81%ID/g at 1 h pi; 6.36 ± 0.85%ID/g at 4 h pi, and 0.41 ± 0.07%ID/g at 4 h pi block). Thus, human GRP-based radioligands, such as [(99m)Tc]Demomedin C, can successfully target GRPR-expressing human tumors in vivo while displaying attractive biological features--e.g. higher GRPR-selectivity--vs their frog-homologues.

Unexpected sensitivity of sst2 antagonists to N-terminal radiometal modifications

Chelated somatostatin agonists have been shown to be sensitive to N-terminal radiometal modifications, with Ga-DOTA agonists having significantly higher binding affinity than their Lu-, In-, and Y-DOTA correlates. Recently, somatostatin antagonists have been successfully developed as alternative tracers to agonists. The aim of this study was to evaluate whether chelated somatostatin antagonists are also sensitive to radiometal modifications and how. We have synthesized 3 different somatostatin antagonists, DOTA-p-NO(2)-Phe-c[D-Cys-Tyr-D-Aph(Cbm)-Lys-Thr-Cys]-D-Tyr-NH(2), DOTA-Cpa-c[D-Cys-Aph(Hor)-D-Aph(Cbm)-Lys-Thr-Cys]-D-Tyr-NH(2) (DOTA-JR11), and DOTA-p-Cl-Phe-c[D-Cys-Tyr-D-Aph(Cbm)-Lys-Thr-Cys]-D-Tyr-NH(2), and added various radiometals including In(III), Y(III), Lu(III), Cu(II), and Ga(III). We also replaced DOTA with 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA) and added Ga(III). The binding affinity of somatostatin receptors 1 through 5 was evaluated in all cases. In all 3 resulting antagonists, the Ga-DOTA analogs were the lowest-affinity radioligands, with a somatostatin receptor 2 binding affinity up to 60 times lower than the respective Y-DOTA, Lu-DOTA, or In-DOTA compounds. Interestingly, however, substitution of DOTA by the NODAGA chelator was able to increase massively its binding affinity in contrast to the Ga-DOTA analog. The 3 NODAGA analogs are antagonists in functional tests. In vivo biodistribution studies comparing (68)Ga-DOTATATE agonist with (68)Ga-DOTA-JR11 and (68)Ga-NODAGA-JR11 showed not only that the JR11 antagonist radioligands were superior to the agonist ligands but also that (68)Ga-NODAGA-JR11 was the tracer of choice and preferable to (68)Ga-DOTA-JR11 in transplantable HEK293-hsst(2) tumors in mice. One may therefore generalize that somatostatin receptor 2 antagonists are sensitive to radiometal modifications and may preferably be coupled with a (68)Ga-NODAGA chelator-radiometal complex.

[111In-DOTA]Somatostatin-14 analogs as potential pansomatostatin-like radiotracers - first results of a preclinical study

In this study, we report on the synthesis, radiolabeling, and biological evaluation of two new somatostatin-14 (SS14) analogs, modified with the universal chelator DOTA. We were interested to investigate if and to what extent such radiotracer prototypes may be useful for targeting sst1-5-expressing tumors in man but, most importantly, to outline potential drawbacks and benefits associated with their use.

The Influence of Different Metal-Chelators on the Biological Profil of Nanoparticles for Gallium-68 Based Molecular Imaging

The use of metal chelators is becoming increasingly important in the development of new tracers for molecular imaging. With the rise of the field of nanotechnology, the fusion of both technologies has shown great potential for clinical applications. The pharmacokinetcs of nanoparticles can be monitored via positron emission tomography (PET) after surface modification and radiolabeling with positron emitting radionuclides. Different metal ion chelators can be used to facilitate labeling of the radionuclides and as a prerequisite, optimized radiolabeling procedure is necessary to prevent nanoparticle aggregation and degradation. However, the effects of chelator modification on nanoparticle pharmacokinetic properties have not been well studied and currently no studies to date have compared the biological effects of the use of different chelators in the surface modification of nanoparticles.

Radiolabeled somatostatin receptor antagonists are preferable to agonists for in vivo peptide receptor targeting of tumors

Targeting neuroendocrine tumors expressing somatostatin receptor subtypes (sst) with radiolabeled somatostatin agonists is an established diagnostic and therapeutic approach in oncology. While agonists readily internalize into tumor cells, permitting accumulation of radioactivity, radiolabeled antagonists do not, and they have not been considered for tumor targeting. The macrocyclic chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was coupled to two potent somatostatin receptor-selective peptide antagonists [NH(2)-CO-c(DCys-Phe-Tyr-DAgl(8)(Me,2-naphthoyl)-Lys-Thr-Phe-Cys)-OH (sst(3)-ODN-8) and a sst(2)-selective antagonist (sst(2)-ANT)], for labeling with (111/nat)In. (111/nat)In-DOTA-sst(3)-ODN-8 and (111/nat)In-DOTA-[4-NO(2)-Phe-c(DCys-Tyr-DTrp-Lys-Thr-Cys)-DTyr-NH(2)] ((111/nat)In-DOTA-sst(2)-ANT) showed high sst(3)- and sst(2)-binding affinity, respectively. They did not trigger sst(3) or sst(2) internalization but prevented agonist-stimulated internalization. (111)In-DOTA-sst(3)-ODN-8 and (111)In-DOTA-sst(2)-ANT were injected intravenously into mice bearing sst(3)- and sst(2)-expressing tumors, and their biodistribution was monitored. In the sst(3)-expressing tumors, strong accumulation of (111)In-DOTA-sst(3)-ODN-8 was observed, peaking at 1 h with 60% injected radioactivity per gram of tissue and remaining at a high level for >72 h. Excess of sst(3)-ODN-8 blocked uptake. As a control, the potent agonist (111)In-DOTA-[1-Nal(3)]-octreotide, with strong sst(3)-binding and internalization properties showed a much lower and shorter-lasting uptake in sst(3)-expressing tumors. Similarly, (111)In-DOTA-sst(2)-ANT was injected into mice bearing sst(2)-expressing tumors. Tumor uptake was considerably higher than with the highly potent sst(2)-selective agonist (111)In-diethylenetriaminepentaacetic acid-[Tyr(3),Thr(8)]-octreotide ((111)In-DTPA-TATE). Scatchard plots showed that antagonists labeled many more sites than agonists. Somatostatin antagonist radiotracers therefore are preferable over agonists for the in vivo targeting of sst(3)- or sst(2)-expressing tumors. Antagonist radioligands for other peptide receptors need to be evaluated in nuclear oncology as a result of this paradigm shift.