A Structure-Activity Relationship Study of Bimodal BODIPY-Labeled PSMA-Targeting Bioconjugates

The aim of this study was to identify a high-affinity BODIPY peptidomimetic that targets the prostate-specific membrane antigen (PSMA) as a potential bimodal imaging probe for prostate cancer. For the structure-activity study, several BODIPY (difluoroboron dipyrromethene) derivatives with varying spacers between the BODIPY dye and the PSMA Glu-CO-Lys binding motif were prepared. Corresponding affinities were determined by competitive binding assays in PSMA-positive LNCaP cells. One compound was identified with comparable affinity (IC50=21.5±0.1 nM) to Glu-CO-Lys-Ahx-HBED-CC (PSMA-11) (IC50=18.4±0.2 nM). Radiolabeling was achieved by Lewis-acid-mediated 19F/18F exchange in moderate molar activities (∼0.7 MBq nmol−1) and high radiochemical purities (>99 %) with mean radiochemical yields of 20–30 %. Cell internalization of the 18F-labeled high-affinity conjugate was demonstrated in LNCaP cells showing gradual increasing PSMA-mediated internalization over time. By fluorescence microscopy, localization of the high-affinity BODIPY-PSMA conjugate was found in the cell membrane at early time points and also in subcellular compartments at later time points. In summary, a high-affinity BODIPY-PSMA conjugate has been identified as a suitable candidate for the development of PSMA-specific dual-imaging agents

Proof-of-Concept Study of the NOTI Chelating Platform: Preclinical Evaluation of 64Cu-Labeled Mono- and Trimeric c(RGDfK) Conjugates

Purpose We recently developed a chelating platform based on the macrocycle 1,4,7-triazacyclononane with up to three five-membered azaheterocyclic arms for the preparation of 68Ga- and 64Cu-based radiopharmaceuticals. Based on this platform, the chelator scaffold NOTI-TVA with three additional carboxylic acid groups for bioconjugation was synthesized and characterized. The primary aims of this proof-of-concept study were (1) to evaluate if trimeric radiotracers on the basis of the NOTI-TVA 6 scaffold can be developed, (2) to determine if the additional substituents for bioconjugation at the non-coordinating NH atoms of the imidazole residues of the building block NOTI influence the metal binding properties, and (3) what influence multiple targeting vectors have on the biological performance of the radiotracer. The cyclic RGDfK peptide that specifically binds to the αvß3 integrin receptor was selected as the biological model system. Procedures Two different synthetic routes for the preparation of NOTI-TVA 6 were explored. Three c(RGDfK) peptide residues were conjugated to the NOTI-TVA 6 building block by standard peptide chemistry providing the trimeric bioconjugate NOTI-TVA-c(RGDfK)3 9. Labeling of 9 with [64Cu]CuCl2 was performed manually at pH 8.2 at ambient temperature. Binding affinities of Cu-8, the Cu2+ complex of the previously described monomer NODIA-Me-c(RGDfK) 8, and the trimer Cu-9 to integrin αvß3 were determined in competitive cell binding experiments in the U-87MG cell line. The pharmacokinetics of both 64Cu-labeled conjugates [64Cu]Cu-8 and [64Cu]Cu-9 were determined by small-animal PET imaging and ex vivo biodistribution studies in mice bearing U-87MG xenografts. Results Depending on the synthetic route, NOTI-TVA 6 was obtained with an overall yield up to 58 %. The bioconjugate 9 was prepared in 41 % yield. Both conjugates [64Cu]Cu-8 and [64Cu]Cu-9 were radiolabeled quantitatively at ambient temperature in high molar activities of Am ~ 20 MBq nmol−1 in less than 5 min. Competitive inhibitory constants IC50 of c(RDGfK) 7, Cu-8, and Cu-9 were determined to be 159.5 ± 1.3 nM, 256.1 ± 2.1 nM, and 99.5 ± 1.1 nM, respectively. In small-animal experiments, both radiotracers specifically delineated αvß3 integrin-positive U-87MG tumors with low uptake in non-target organs and rapid blood clearance. The trimer [64Cu]Cu-9 showed a ~ 2.5-fold higher tumor uptake compared with the monomer [64Cu]Cu-8. Conclusions Functionalization of NOTI at the non-coordinating NH atoms of the imidazole residues for bioconjugation was straightforward and allowed the preparation of a homotrimeric RGD conjugate. After optimization of the synthesis, required building blocks to make NOTI-TVA 6 are now available on multi-gram scale. Modifications at the imidazole groups had no measurable impact on metal binding properties in vitro and in vivo suggesting that the NOTI scaffold is a promising candidate for the development of 64Cu-labeled multimeric/multifunctional radiotracers

In vitro and in vivo evaluation of the bifunctional chelator NODIA-Me in combination with a prostate-specific membrane antigen targeting vector

Introduction We recently developed a chelating platform based on the macrocycle 1,4,7-triazacyclononane with up to three five-membered azaheterocyclic arms for complexation of the PET nuclides gallium-68 and copper-64. The main objective of this study was to evaluate the stability and pharmacokinetics of 68Ga- and 64Cu-complexes of the bifunctional chelator NODIA-Me 1 covalently bound to a PSMA targeting vector in vivo. Methods NODIA-Me 1 was conjugated to the PSMA targeting Glu-NH-CO-NH-Lys moiety to give the bioconjugate NODIA-Me-NaI-Ahx-PSMA 4. The stability of [68Ga]4 and [64Cu]4 was assessed in vitro by serum stability studies. The PSMA binding affinity was determined in competitive cell experiments in LNCaP cells using 68Ga-PSMA-HBED-CC as radioligand. The stability and pharmacokinetics of [68Ga]4 and [64Cu]4 was evaluated by PET imaging and ex vivo biodistribution studies in mice bearing subcutaneous LNCaP tumors. Results In human serum, [68Ga]4 and [64Cu]4 remained intact to 85% (3 h) and 92% (24 h), respectively. Nature of the metal chelate influenced PSMA binding affinity with IC50 of 233 ± 10 nM for uncomplexed 4, 681 ± 7 nM for Cu-4 and 176 ± 10 nM for Ga-4. In animal studies, [68Ga]4 and [64Cu]4 revealed low uptake (≤1% IA g−1) in the majority of organs. Kidney uptake at 1 h p.i. was 6.28 ± 0.92% IA g−1 and 4.96 ± 0.79% IA g−1 and specific tumor uptake was 1.33 ± 0.46% IA g−1 and 2.15 ± 0.38% IA g−1 for [68Ga]4 and [64Cu]4, respectively. Conclusion The bifunctional chelator NODIA-Me 1 was successfully conjugated to a PSMA targeting moiety. In small-animal PET imaging and ex vivo biodistribution studies, 68Ga- and 64Cu-labelled conjugates specifically delineated PSMA-positive LNCaP tumors and exhibited rapid renal clearance from non-target tissues with no significant demetallation/transchelation in vivo. The results support further development of this novel chelating platform for production of 68Ga- and 64Cu-labelled radiopharmaceuticals

Preparation and preclinical evaluation of a 68Ga-labelled c(RGDfK) conjugate comprising the bifunctional chelator NODIA-Me

Abstract Background We recently developed a chelating platform based on the macrocycle 1,4,7-triazacyclononane with up to three, five-membered azaheterocyclic arms for the development of 68Ga- and 64Cu-based radiopharmaceuticals. Here, a 68Ga-labelled conjugate comprising the bifunctional chelator NODIA-Me in combination with the αvß3-targeting peptide c(RGDfK) has been synthesized and characterized. The primary aim was to evaluate further the potential of our NODIA-Me chelating system for the development of 68Ga-labelled radiotracers. Results The BFC NODIA-Me was conjugated to c(RGDfK) by standard peptide chemistry to obtain the final bioconjugate NODIA-Me-c(RGDfK) 3 in 72% yield. Labelling with [68Ga]GaCl3 was accomplished in a fully automated, cGMP compliant process to give [68Ga]3 in high radiochemical yield (98%) and moderate specific activity (~ 8 MBq nmol− 1). Incorporation of the Ga-NODIA-Me chelate to c(RGDfK) 2 had only minimal influence on the affinity to integrin αvß3 (IC50 values [natGa]3 = 205.1 ± 1.4 nM, c(RGDfK) 2 = 159.5 ± 1.3 nM) as determined in competitive cell binding experiments in U-87 MG cell line. In small-animal PET imaging and ex vivo biodistribution studies, the radiotracer [68Ga]3 showed low uptake in non-target organs and specific tumor uptake in U-87 MG tumors. Conclusion The results suggest that the bifunctional chelator NODIA-Me is an interesting alternative to existing ligands for the development of 68Ga-labelled radiopharmaceuticals

In vitro and in vivo evaluation of the bifunctional chelator NODIA-Me in combination with a prostate-specific membrane antigen targeting vector

INTRODUCTION: We recently developed a chelating platform based on the macrocycle 1,4,7-triazacyclononane with up to three five-membered azaheterocyclic arms for complexation of the PET nuclides gallium-68 and copper-64. The main objective of this study was to evaluate the stability and pharmacokinetics of 68Ga- and 64Cu-complexes of the bifunctional chelator NODIA-Me 1 covalently bound to a PSMA targeting vector in vivo. METHODS: NODIA-Me 1 was conjugated to the PSMA targeting Glu-NH-CO-NH-Lys moiety to give the bioconjugate NODIA-Me-NaI-Ahx-PSMA 4. The stability of [68Ga]4 and [64Cu]4 was assessed in vitro by serum stability studies. The PSMA binding affinity was determined in competitive cell experiments in LNCaP cells using 68Ga-PSMA-HBED-CC as radioligand. The stability and pharmacokinetics of [68Ga]4 and [64Cu]4 was evaluated by PET imaging and ex vivo biodistribution studies in mice bearing subcutaneous LNCaP tumors. RESULTS: In human serum, [68Ga]4 and [64Cu]4 remained intact to 85% (3 h) and 92% (24 h), respectively. Nature of the metal chelate influenced PSMA binding affinity with IC50 of 233 ± 10 nM for uncomplexed 4, 681 ± 7 nM for Cu-4 and 176 ± 10 nM for Ga-4. In animal studies, [68Ga]4 and [64Cu]4 revealed low uptake (≤1% IA g-1) in the majority of organs. Kidney uptake at 1 h p.i. was 6.28 ± 0.92% IA g-1 and 4.96 ± 0.79% IA g-1 and specific tumor uptake was 1.33 ± 0.46% IA g-1 and 2.15 ± 0.38% IA g-1 for [68Ga]4 and [64Cu]4, respectively. CONCLUSION: The bifunctional chelator NODIA-Me 1 was successfully conjugated to a PSMA targeting moiety. In small-animal PET imaging and ex vivo biodistribution studies, 68Ga- and 64Cu-labelled conjugates specifically delineated PSMA-positive LNCaP tumors and exhibited rapid renal clearance from non-target tissues with no significant demetallation/transchelation in vivo. The results support further development of this novel chelating platform for production of 68Ga- and 64Cu-labelled radiopharmaceuticals

Identification, Characterization, and Suppression of Side Products Formed during the Synthesis of [177Lu]Lu-PSMA-617

In recent years, radiolabeled tracers targeting prostate-specific membrane antigen (PSMA) have had a tremendous impact on prostate cancer management. Here, we report on the formation of radioactive impurities formed during the clinical production of 177Lu-labeled PSMA-617. We provide compelling evidence that these impurities are the result of a spontaneous, thermally mediated condensation reaction of the Glu-CO-Lys moiety resulting in the formation of three different five-membered ring systems. Density functional theory (DFT) calculations show that the condensation and cyclization of the Glu-CO-Lys moiety is thermodynamically spontaneous. In cell experiments, no affinity of these cyclized compounds toward PSMA was observed. HPLC analyses of urine samples from patient studies showed rapid renal excretion of these radioactive cyclized species. Radiolabeling conditions were identified that significantly reduced the formation of cyclized side products yielding 177Lu-labeled PSMA-617 in high radiochemical yield and purity in concordance with current good manufacturing practice (cGMP) requirements

Optimized synthesis and indium complex formation with the bifunctional chelator NODIA-Me

The bifunctional chelator NODIA-Me holds promise for radiopharmaceutical development. NODIA-Me is based on the macrocycle TACN (1,4,7-triazacyclononane) and incorporates two additional methylimidazole arms for metal chelation and an acetic acid residue for bioconjugation. The original two step synthesis was less than optimal due to low yields and the requirement of semi-preparative RP-HPLC purifications. Here, the overall yield for the preparation of NODIA-Me was improved two- to five-fold via two synthetic routes using different protection/deprotection techniques. This way, it was possible (1) to prepare of NODIA-Me on multi-gram scale and (2) to avoid time-consuming HPLC purifications. Inspired by recent results with nat/68Ga3+, preliminary studies on the radiolabeling properties and complex formation of NODIA-Me with nat/111In3+ were performed. Quantitative radiochemical yields were achieved at ambient temperature providing molar activities of ∼30 MBq nmol-1, which could be increased to ∼240 MBq nmol-1 at 95 °C. At r.t., pH 5.5 was optimal for 111In-labeling, but quantitative yields were also achieved in the pH range from 5.5 to 8.2, when the reaction temperature was increased. Stability tests of 111In complexes in vitro revealed high kinetic stabilities in serum and ligand challenge experiments, which is a consequence of the formation of rigid 1 : 1 indium chelates as shown by NMR studies in solution. In summary, the new synthetic routes afford the BFC NODIA-Me in high yields and on large scale. Further, 111In complexation experiments broaden the scope of our chelating system for radiopharmaceutical applications

Optimized synthesis and indium complex formation with the bifunctional chelator NODIA-Me

The bifunctional chelator NODIA-Me holds promise for radiopharmaceutical development. NODIA-Me is based on the macrocycle TACN (1,4,7-triazacyclononane) and incorporates two additional methylimidazole arms for metal chelation and an acetic acid residue for bioconjugation. The original two step synthesis was less than optimal due to low yields and the requirement of semi-preparative RP-HPLC purifications. Here, the overall yield for the preparation of NODIA-Me was improved two- to five-fold via two synthetic routes using different protection/deprotection techniques. This way, it was possible (1) to prepare of NODIA-Me on multi-gram scale and (2) to avoid time-consuming HPLC purifications. Inspired by recent results with nat/68Ga3+, preliminary studies on the radiolabeling properties and complex formation of NODIA-Me with nat/111In3+ were performed. Quantitative radiochemical yields were achieved at ambient temperature providing molar activities of ∼30 MBq nmol−1, which could be increased to ∼240 MBq nmol−1 at 95 °C. At r.t., pH 5.5 was optimal for 111In-labeling, but quantitative yields were also achieved in the pH range from 5.5 to 8.2, when the reaction temperature was increased. Stability tests of 111In complexes in vitro revealed high kinetic stabilities in serum and ligand challenge experiments, which is a consequence of the formation of rigid 1 : 1 indium chelates as shown by NMR studies in solution. In summary, the new synthetic routes afford the BFC NODIA-Me in high yields and on large scale. Further, 111In complexation experiments broaden the scope of our chelating system for radiopharmaceutical applications

Salt Marsh Accretion and Storm Tide Variation: an Example from a Barrier Island in the North Sea

We reconstruct past accretion rates of a salt marsh on the island of Sylt, Germany, using measurements of the radioisotopes 210Pb and 137Cs, as well as historical aerial photographs. Results from three cores indicate accretion rates varying between 1 and 16 mm year−1. Comparisons with tide gauge data show that high accretion rates during the 1980s and 1990s coincide with periods of increased storm activity. We identify a critical inundation height of 18 cm below which the strength of a storm seems to positively influence salt marsh accretion rates and above which the frequency of storms becomes the major factor. In addition to sea level rise, we conclude that in low marsh zones subject to higher inundation levels, mean storm strength is the major factor affecting marsh accretion, whereas in high marsh zones with lower inundation levels, it is storm frequency that impacts marsh accretion