Human ABC and SLC Transporters: The Culprit Responsible for Unspecific PSMA-617 Uptake?

[177Lu]Lu-PSMA-617 has recently been successfully approved by the FDA, the MHRA, Health Canada and the EMA as Pluvicto®. However, salivary gland (SG) and kidney toxicities account for its main dose-limiting side-effects, while its corresponding uptake and retention mechanisms still remain elusive. Recently, the presence of different ATP-binding cassette (ABC) transporters, such as human breast cancer resistance proteins (BCRP), multidrug resistance proteins (MDR1), multidrug-resistance-related proteins (MRP1, MRP4) and solute cassette (SLC) transporters, such as multidrug and toxin extrusion proteins (MATE1, MATE2-K), organic anion transporters (OAT1, OAT2v1, OAT3, OAT4) and peptide transporters (PEPT2), has been verified at different abundances in human SGs and kidneys. Therefore, our aim was to assess whether [177Lu]Lu-PSMA-617 and [225Ac]Ac-PSMA-617 are substrates of these ABC and SLC transporters. For in vitro studies, the novel isotopologue ([α,ß-3H]Nal)Lu-PSMA-617 was used in cell lines or vesicles expressing the aforementioned human ABC and SLC transporters for inhibition and uptake studies, respectively. The corresponding probe substrates and reference inhibitors were used as controls. Our results indicate that [177Lu]Lu-PSMA-617 and [225Ac]Ac-PSMA-617 are neither inhibitors nor substrates of the examined transporters. Therefore, our results show that human ABC and SLC transporters play no central role in the uptake and retention of [177Lu]Lu-PSMA-617 and [225Ac]Ac-PSMA-617 in the SGs and kidneys nor in the observed toxicities.

Synthesis of tritium-labeled Lu-PSMA-617: Alternative tool for biological evaluation of radiometal-based pharmaceuticals.

This project focuses on the generation and evaluation of functional alternatives to radiometal-based pharmaceuticals supporting basic research and the in vitro developmental phase. Employing robust tritium chemistry and non-radioactive metal surrogates in two synthetic and labeling strategies resulted in ([ring-3H]Nal)PSMA-617 and ([α,ß-3H]Nal)PSMA-617. In particular, ([α,ß-3H]Nal)Lu-PSMA-617 exhibited high radiolytic as well as metal-complex stability and was compared to the clinically-established radiopharmaceutical [177Lu]Lu-PSMA-617. The cell-based assays confirmed the applicability of ([α,ß-3H]Nal)Lu-PSMA-617 as a substitute of [177Lu]Lu-PSMA-617 in pre-clinical biological settings.

Development of PSMA-1007-Related Series of 18F-Labeled Glu-Ureido-Type PSMA Inhibitors.

In recent years, a number of drugs targeting the prostate-specific membrane antigen (PSMA) have become important tools in the diagnosis and treatment of prostate cancer. In the present work, we report on the synthesis and preclinical evaluation of a series of 18F-labeled PSMA ligands for diagnostic application based on the theragnostic ligand PSMA-617. By applying modifications to the linker structure, insight into the structure-activity relationship could be gained, highlighting the importance of hydrophilicity and stereoselectivity on interaction with PSMA and hence the biodistribution. Selected compounds were co-crystallized with the PSMA protein and analyzed by X-rays with mixed results. Among these, PSMA-1007 (compound 5) showed the best interaction with the PSMA protein. The respective radiotracer [18F]PSMA-1007 was translated into the clinic and is, in the meantime, subject of advanced clinical trials.

HBED-NN: A Bifunctional Chelator for Constructing Radiopharmaceuticals.

Radiometal-based radiopharmaceuticals bearing bifunctional HBED chelators are powerful radiotracers for cancer diagnosis and therapy. Bifunctional HBED chelators make strong complexes with trivalent gallium and are able to bind to bioactive molecules through covalent bonds. However, thus far, no bifunctional HBED chelator capable of direct conjugation via click chemistry has been reported. We hereby introduce HBED-NN as a structurally new bifunctional HBED chelator for direct click coupling. We also investigated the complex chemistry of [Ga-(HBED-NN)] for potential use in gallium-based radiopharmaceuticals.

Designing tracers for PET imaging of the urokinase-type plasminogen activator receptor from a cyclic uPA-derived peptide: first in vitro evaluations.

The treatment of cancer remains a major challenge, especially after tumour cell dissemination and metastases formation. Expression of the urokinase-type plasminogen activation system including urokinase (uPA) and its receptor (uPAR) has been associated with the complex process of cell migration, a tumour's invasive potential as well as a reduced overall and disease-free survival of patients with solid cancers and haematological disorders. A cyclic peptide cyclo[21,29][d-Cys21 ,Cys29 ]-uPA21-30 was designed from the growth factor-like domain (GFD) of urokinase whose binding to uPAR was found to inhibit tumour growth and spread of human ovarian cancer cells in mice. With the aim of visualising uPAR expression using PET imaging to attempt an estimate on the tumour's aggressiveness, the cyclic peptide was modified with an either C- or N-terminally attached variable spacer and chelator. The free ligands were evaluated for their binding affinities to the isolated human uPAR and labelled with 68 Ga and 177 Lu to assess their lipophilicities and stabilities in human serum. Although retaining the full binding potential displayed by cyclo[21,29][d-Cys21 ,Cys29 ]-uPA21-30 to its target was found to be a challenging task upon both C- and N-terminal modification, chelator-bearing ligands were identified that can serve as promising starting points in the development of uPAR-addressing PET tracers.

Procedures for the GMP-Compliant Production and Quality Control of [18F]PSMA-1007: A Next Generation Radiofluorinated Tracer for the Detection of Prostate Cancer.

Radiolabeled tracers targeting the prostate-specific membrane antigen (PSMA) have become important radiopharmaceuticals for the PET-imaging of prostate cancer. In this connection, we recently developed the fluorine-18-labelled PSMA-ligand [18F]PSMA-1007 as the next generation radiofluorinated Glu-ureido PSMA inhibitor after [18F]DCFPyL and [18F]DCFBC. Since radiosynthesis so far has been suffering from rather poor yields, novel procedures for the automated radiosyntheses of [18F]PSMA-1007 have been developed. We herein report on both the two-step and the novel one-step procedures, which have been performed on different commonly-used radiosynthesisers. Using the novel one-step procedure, the [18F]PSMA-1007 was produced in good radiochemical yields ranging from 25 to 80% and synthesis times of less than 55 min. Furthermore, upscaling to product activities up to 50 GBq per batch was successfully conducted. All batches passed quality control according to European Pharmacopoeia standards. Therefore, we were able to disclose a new, simple and, at the same time, high yielding production pathway for the next generation PSMA radioligand [18F]PSMA-1007. Actually, it turned out that the radiosynthesis is as easily realised as the well-known [18F]FDG synthesis and, thus, transferable to all currently-available radiosynthesisers. Using the new procedures, the clinical daily routine can be sustainably supported in-house even in larger hospitals by a single production batch.

Novel Preclinical and Radiopharmaceutical Aspects of [68Ga]Ga-PSMA-HBED-CC: A New PET Tracer for Imaging of Prostate Cancer.

The detection of prostate cancer lesions by PET imaging of the prostate-specific membrane antigen (PSMA) has gained highest clinical impact during the last years. 68Ga-labelled Glu-urea-Lys(Ahx)-HBED-CC ([68Ga]Ga-PSMA-HBED-CC) represents a successful novel PSMA inhibitor radiotracer which has recently demonstrated its suitability in individual first-in-man studies. The radiometal chelator HBED-CC used in this molecule represents a rather rarely used acyclic complexing agent with chemical characteristics favourably influencing the biological functionality of the PSMA inhibitor. The simple replacement of HBED-CC by the prominent radiometal chelator DOTA was shown to dramatically reduce the in vivo imaging quality of the respective 68Ga-labelled PSMA-targeted tracer proving that HBED-CC contributes intrinsically to the PSMA binding of the Glu-urea-Lys(Ahx) pharmacophore. Owing to the obvious growing clinical impact, this work aims to reflect the properties of HBED-CC as acyclic radiometal chelator and presents novel preclinical data and relevant aspects of the radiopharmaceutical production process of [68Ga]Ga-PSMA-HBED-CC.