It’s Time to Shift the Paradigm: Translation and Clinical Application of Non-αvβ3 Integrin Targeting Radiopharmaceuticals

For almost the entire period of the last two decades, translational research in the area of integrin-targeting radiopharmaceuticals was strongly focused on the subtype αvβ3, owing to its expression on endothelial cells and its well-established role as a biomarker for, and promoter of, angiogenesis. Despite a large number of translated tracers and clinical studies, a clinical value of αvβ3-integrin imaging could not be defined yet. The focus of research has, thus, been moving slowly but steadily towards other integrin subtypes which are involved in a large variety of tumorigenic pathways. Peptidic and non-peptidic radioligands for the integrins α5β1, αvβ6, αvβ8, α6β1, α6β4, α3β1, α4β1, and αMβ2 were first synthesized and characterized preclinically. Some of these compounds, targeting the subtypes αvβ6, αvβ8, and α6β1/β4, were subsequently translated into humans during the last few years. αvβ6-Integrin has arguably attracted most attention because it is expressed by some of the cancers with the worst prognosis (above all, pancreatic ductal adenocarcinoma), which substantiates a clinical need for the respective theranostic agents. The receptor furthermore represents a biomarker for malignancy and invasiveness of carcinomas, as well as for fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF), and probably even for Sars-CoV-2 (COVID-19) related syndromes. Accordingly, the largest number of recent first-in-human applications has been reported for radiolabeled compounds targeting αvβ6-integrin. The results indicate a substantial clinical value, which might lead to a paradigm change and trigger the replacement of αvβ3 by αvβ6 as the most popular integrin in theranostics.

Radiolabeled Compounds for Diagnosis and Treatment of Cancer

Nuclear medicine was recognized as a potential medical field a long time ago when 131I was used in thyroid cancer patients [...]

Novel CYP11B-ligand [123/131I]IMAZA as promising theranostic tool for adrenocortical tumors: comprehensive preclinical characterization and first clinical experience

Purpose Adrenal tumors represent a diagnostic and therapeutic challenge. Promising results have been obtained through targeting the cytochrome P450 enzymes CYP11B1 and CYP11B2 for molecular imaging, and [123/131I]iodometomidate ([123/131I]IMTO) has even been successfully introduced as a theranostic agent. As this radiopharmaceutical shows rapid metabolic inactivation, we aimed at developing new improved tracers. Methods Several IMTO derivatives were newly designed by replacing the unstable methyl ester by different carboxylic esters or amides. The inhibition of aldosterone and cortisol synthesis was tested in different adrenocortical cell lines. The corresponding radiolabeled compounds were assessed regarding their stability, in vitro cell uptake, in vivo biodistribution in mice, and their binding specificity to cryosections of human adrenocortical and non-adrenocortical tissue. Furthermore, a first investigation was performed in patients with known metastatic adrenal cancer using both [123I]IMTO and the most promising compound (R)-1-[1-(4-[123I]iodophenyl)ethyl]-1H-imidazole-5-carboxylic acid azetidinylamide ([123I]IMAZA) for scintigraphy. Subsequently, a first endoradiotherapy with [131I]IMAZA in one of these patients was performed. Results We identified three analogues to IMTO with high-affinity binding to the target enzymes and comparable or higher metabolic stability and very high and specific accumulation in adrenocortical cells in vitro and in vivo. Labeled IMAZA exhibited superior pharmacokinetic and imaging properties compared to IMTO in mice and 3 patients, too. An endoradiotherapy with [131I]IMAZA induced a 21-month progression-free interval in a patient with rapidly progressing ACC prior this therapy. Conclusion We developed the new radiopharmaceutical [123/131I]IMAZA with superior properties compared to the reference compound IMTO and promising first experiences in humans.

DoE Optimization Empowers the Automated Preparation of Enantiomerically Pure [18F]Talazoparib and its In Vivo Evaluation as a PARP Radiotracer.

PARP inhibitors are proven chemotherapeutics and serve as lead structures for the development of PARP-targeted in vivo imaging probes. Given the clinical potential of PARP imaging for the detection and stratification of various cancers, the development of novel PARP imaging probes with improved pharmacologi-cal profiles over established PARP imaging agents is warranted. Here, we present a novel 18F-labeled PARP radiotracer based on the clinically superior PARP inhibitor talazoparib. An automated radiosynthesis of [18F]talazoparib (RCY: 13 ± 3.4 %; n = 4; molar radioactivity 52 – 176 GBq/μmol) was achieved using a “Design of Experiments” (DoE) optimized copper-mediated radiofluorination reaction. The chiral product was isolated from the reaction mixture using 2D reversed-phase/chiral radio-HPLC (>99% ee). (8S, 9R)-[18F]Talazoparib demonstrated PARP binding in HCC1937 cells in vitro and showed an excellent tumor-to-blood ratio in xeno-graft-bearing mice (10.2 ± 1.5). Despite expected uptake into muscle, bone, and abdominal tissue, a favorable pharmacological profile in terms of excretion, blood half-life, and target engagement was observed in the pilot in vivo study. This synthesis of [18F]talazoparib exemplifies how a DoE based tracer development pipeline can enable the radiosyntheses of clinically relevant but synthetically challenging radiolabeled compounds of high interest to the imaging community.

DoE Optimization Empowers the Automated Preparation of Enantiomerically Pure [18F]Talazoparib and its In Vivo Evaluation as a PARP Radiotracer.

PARP inhibitors are proven chemotherapeutics and serve as lead structures for the development of PARP-targeted in vivo imaging probes. Given the clinical potential of PARP imaging for the detection and stratification of various cancers, the development of novel PARP imaging probes with improved pharmacologi-cal profiles over established PARP imaging agents is warranted. Here, we present a novel 18F-labeled PARP radiotracer based on the clinically superior PARP inhibitor talazoparib. An automated radiosynthesis of [18F]talazoparib (RCY: 13 ± 3.4 %; n = 4; molar radioactivity 52 – 176 GBq/μmol) was achieved using a “Design of Experiments” (DoE) optimized copper-mediated radiofluorination reaction. The chiral product was isolated from the reaction mixture using 2D reversed-phase/chiral radio-HPLC (>99% ee). (8S, 9R)-[18F]Talazoparib demonstrated PARP binding in HCC1937 cells in vitro and showed an excellent tumor-to-blood ratio in xeno-graft-bearing mice (10.2 ± 1.5). Despite expected uptake into muscle, bone, and abdominal tissue, a favorable pharmacological profile in terms of excretion, blood half-life, and target engagement was observed in the pilot in vivo study. This synthesis of [18F]talazoparib exemplifies how a DoE based tracer development pipeline can enable the radiosyntheses of clinically relevant but synthetically challenging radiolabeled compounds of high interest to the imaging community.

An efficient and label-free LC-MS/MS method for assessing drug’s activity at dopamine and serotonin transporters using transporter-transfected HEK293T cells

Background: Dopamine transporter (DAT) and serotonin transporter (SERT) are targets for many psychoactive substances. Functional assays including uptake inhibition and release assays often involve radiolabeled compounds like [3H]-dopamine and [3H]-serotonin to assess drug activity at transporters, which have high requirements on handling radioactive samples. Aims: The aim of this study was to establish a label-free method to assess drug activity at DAT and SERT. Methods: A liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was established using transporter-transfected human embryonic kidney 293T (HEK293T) cells. This method was evaluated by testing the effects of amphetamine and cocaine in the assay procedure. Results: The limits of detection of this method were 0.2 nM for both dopamine (DA) and serotonin (5-HT), with good linearities in the range of 0.5–160 nM. Amphetamine and cocaine’s IC50 and EC50 on DAT and SERT determined by this method were consistent with previous reports. Conclusions: A rapid, reliable and label-free LC-MS/MS method for assessing drug activity was established, which affords an attractive alternative for those laboratories that do not have a radiation license or capabilities.

Fast Fluorine-18 labeling and preclinical evaluation of novel Mucin1 and its Folate hybrid peptide conjugate for targeting breast carcinoma

Background There is a need to develop new and more potent radiofluorinated peptide and their hybrid conjugates for multiple-receptors targeting properties that overexpress on many cancers. Methods We have synthesized MUC1-[18F] SFB and MUC1-FA-[18F] SFB hybrid conjugates using a convenient and one-step nucleophilic displacement reaction. In vitro cell binding and in vivo evaluation in animals were performed to determine the potential of these radiolabeled compounds. Results Radiochemical yields for MUC1-[18F] SFB and MUC1-FA-[18F] SFB conjugates were greater than 70% in less than 30 min synthesis time. Radiochemical purities were greater than 97% without HPLC purification, which makes these approaches amenable to automation. In vitro studies on MCF7 breast cancer cells showed that the significant amounts of the radiofluorinated conjugates were associated with cell fractions and held good affinity and specificity for MCF7 cells. In vivo characterization in Balb/c mice revealed rapid blood clearance with excretion predominantly by urinary as well as hepatobiliary systems for MUC1-[18F] SFB and MUC1-FA-[18F] SFB, respectively. Biodistribution in SCID mice bearing MCF7 xenografts, demonstrated excellent tumor uptake (12% ID/g) and favorable kinetics for MUC1-FA-[18F] SFB over MUC1-[18F]SFB. The tumor uptake was blocked by the excess co-injection of cold peptides suggesting the receptor-mediated process. Conclusion Initial PET/CT imaging of SCID mice with MCF7 xenografts, confirmed these observations. These results demonstrate that MUC1-FA-[18F] SFB may be a useful PET imaging probe for breast cancer detection and monitoring tumor response to the treatment.

Amyloid β-binding Bifunctional Chelators with Favorable Lipophilicity for 64Cu PET Imaging in Alzheimer’s Disease

Herein we report a new series of bifunctional chelators (BFCs) with high affinity for amyloid aggregates, strong binding affinity towards Cu(II) and favorable lipophilicity for potential blood-brain barrier (BBB) penetration. The alkyl carboxylate pendant arms offer up to three orders of magnitude higher binding affinity towards Cu(II) vs. the parent chelating fragment, and can generate fairly stable Cu complexes, including ⁶⁴Cu-radiolabeled compounds. Among the five compounds tested, the ⁶⁴Cu-YW-7 and ⁶⁴Cu-YW-10 complexes exhibit strong and specific staining of amyloid plaques in <i>ex vivo</i> autoradiography studies. Importantly, these compounds have promising partition coefficient (Log D) values of 0.91-1.26 and show moderate brain uptake in biodistribution studies using CD-1 mice. Overall, these BFCs could serve as lead compounds for the development of positron emission tomography (PET) imaging agents for AD diagnosis.

Amyloid β-binding Bifunctional Chelators with Favorable Lipophilicity for 64Cu PET Imaging in Alzheimer’s Disease

Herein we report a new series of bifunctional chelators (BFCs) with high affinity for amyloid aggregates, strong binding affinity towards Cu(II) and favorable lipophilicity for potential blood-brain barrier (BBB) penetration. The alkyl carboxylate pendant arms offer up to three orders of magnitude higher binding affinity towards Cu(II) vs. the parent chelating fragment, and can generate fairly stable Cu complexes, including ⁶⁴Cu-radiolabeled compounds. Among the five compounds tested, the ⁶⁴Cu-YW-7 and ⁶⁴Cu-YW-10 complexes exhibit strong and specific staining of amyloid plaques in <i>ex vivo</i> autoradiography studies. Importantly, these compounds have promising partition coefficient (Log D) values of 0.91-1.26 and show moderate brain uptake in biodistribution studies using CD-1 mice. Overall, these BFCs could serve as lead compounds for the development of positron emission tomography (PET) imaging agents for AD diagnosis.