Discovery of Glutamate Carboxypeptidase III Ligands to Compete the Uptake of [177Lu]Lu-PSMA-617 in Healthy Organs.

Prostate-specific membrane antigen (PSMA)-targeted radio ligand therapeutics (RLTs), such as [177Lu]Lu-PSMA-617 (Pluvicto), have been shown to accumulate in salivary glands and kidneys, potentially leading to undesired side effects. As unwanted accumulation in normal organs may derive from the cross-reactivity of PSMA ligands to glutamate carboxypeptidase III (GCPIII), it may be convenient to block this interaction with GCPIII-selective ligands. Parallel screening of a DNA-encoded chemical library (DEL) against GCPIII and PSMA allowed the identification of GCPIII binders. Structure-activity relationship (SAR) studies resulted in the identification of nanomolar GCPIII ligands with up to 1000-fold selectivity over PSMA. We studied the ability of GCPIII ligands to counteract the binding of [177Lu]Lu-PSMA-617 to human salivary glands by autoradiography and could demonstrate a partial radioprotection.

Unraveling Interaction of Rhenium-188 Microspheres with Primary Hepatic Cancer Cell: A Breakthrough Study.

Introduction: Hepatocellular carcinoma is a prevalent contributor to global mortality rates. The main palliative treatments are trans-arterial chemoembolization and selective intra-arterial radionuclide therapy. Methods: A novel freeze-dried nonradioactive microsphere kit formulation has been developed, and the behavior and therapeutic potential of 188Re microspheres have been assessed. The microspheres were labeled with fluorescein isothiocyanate (FITC) and 188ReO4-. The uptake of FITC microspheres by HepG2 cells was examined at various time intervals. The impact of 188Re microspheres on cell viability and the mode of cell death were investigated with HepG2 cells using MTT and Annexin FITC-V/propidium iodide (PI) apoptosis assay. Results: The labeling efficiency of microspheres was more than 99% with FITC and 188ReO4-. The maximum uptake of FITC microspheres by HepG2 cells was achieved at 6 h. The exposure to 188Re microspheres has shown a decrease in cellular viability from 77.81% ± 0.015% to 42.03% ± 0.148% at 192 h of incubation (∼11 half-lives). The cellular uptake of 188Re microspheres was 0.255-0.901 MBq. These values were concordant with Annexin FITC-V/PI apoptosis assay. At 192 h, 53.28% ± 0.01% of cells entered the apoptotic phase after treatment with 188Re microspheres, and only 39.34% ± 0.02% of cells remained viable. However, in the cells treated with 188ReO4- alone, 74.86% ± 0.005% of cells were viable, and only 24.75% ± 0.577% of cells were in the early apoptotic phase at 192 h. Conclusion: The data revealed that 188Re microspheres treatment led to significant growth inhibition in HepG2 cells compared with 188ReO4-.

Engineering CAR-T cells for radiohapten capture in imaging and radioimmunotherapy applications.

Rationale: The in vivo dynamics of CAR-T cells remain incompletely understood. Novel methods are urgently needed to longitudinally monitor transferred cells non-invasively for biodistribution, functionality, proliferation, and persistence in vivo and for improving their cytotoxic potency in case of treatment failure. Methods: Here we engineered CD19 CAR-T cells ("Thor"-cells) to express a membrane-bound scFv, huC825, that binds DOTA-haptens with picomolar affinity suitable for labeling with imaging or therapeutic radionuclides. We assess its versatile utility for serial tracking studies with PET and delivery of α-radionuclides to enhance anti-tumor killing efficacy in sub-optimal adoptive cell transfer in vivo using Thor-cells in lymphoma models. Results: We show that this reporter gene/probe platform enables repeated, sensitive, and specific assessment of the infused Thor-cells in the whole-body using PET/CT imaging with exceptionally high contrast. The uptake on PET correlates with the Thor-cells on a cellular and functional level. Furthermore, we report the ability of Thor-cells to accumulate cytotoxic alpha-emitting radionuclides preferentially at tumor sites, thus increasing therapeutic potency. Conclusion: Thor-cells are a new theranostic agent that may provide crucial information for better and safer clinical protocols of adoptive T cell therapies, as well as accelerated development strategies.

Fibroblast Activation Protein Inhibitor-Based Radionuclide Therapies: Current Status and Future Directions.

Metastatic malignancies have limited management strategies and variable treatment responses. Cancer cells develop beside and depend on the complex tumor microenvironment. Cancer-associated fibroblasts, with their complex interaction with tumor and immune cells, are involved in various steps of tumorigenesis, such as growth, invasion, metastasis, and treatment resistance. Prooncogenic cancer-associated fibroblasts emerged as attractive therapeutic targets. However, clinical trials have achieved suboptimal success. Fibroblast activation protein (FAP) inhibitor-based molecular imaging has shown encouraging results in cancer diagnosis, making them innovative targets for FAP inhibitor-based radionuclide therapies. This review summarizes the results of preclinical and clinical FAP-based radionuclide therapies. We will describe advances and FAP molecule modification in this novel therapy, as well as its dosimetry, safety profile, and efficacy. This summary may guide future research directions and optimize clinical decision-making in this emerging field.

DNA-PKcs inhibitors sensitize neuroendocrine tumor cells to peptide receptor radionuclide therapy in vitro and in vivo.

Background: Peptide receptor radionuclide therapy (PRRT) increases progression-free survival and quality of life of neuroendocrine tumor (NET) patients, however complete cures are rare and dose-limiting toxicity has been reported. PRRT induces DNA damage of which DNA double strand breaks (DSBs) are the most cytotoxic. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a key player in DSB repair and its inhibition therefore is a potential way to enhance PRRT efficacy without increasing the dosage. Methods: We analyzed effects of combining PRRT and DNA-PKcs inhibitor AZD7648 on viability, cell death and clonogenic survival on SSTR2-expressing cell lines BON1-SSTR2, GOT1 and NCI-H69. Therapy-induced DNA damage response was assessed by analyzing DSB foci levels and cell cycle distributions. In vivo efficacy was investigated in BON1-SSTR2 and NCI-H69 xenografted mice and hematologic and renal toxicity were monitored by blood counts, creatinine levels and analyzing renal morphology. Results: Combining PRRT and AZD7648 significantly decreased viability of BON1-SSTR2, GOT1 and NCI-H69 cells and induced cell death in GOT1 and BON1-SSTR2 cells. A strong effect of AZD7648 on PRRT-induced DSB repair was found. In GOT1 cells, this was accompanied by induction of cell cycle blocks. However, BON1-SSTR2 cells were unable to fully arrest their cell cycle and polyploid cells with high DNA damage levels were detected. In vivo, AZD7648 significantly sensitized BON1-SSTR2 and NCI-H69 xenograft models to PRRT. In addition, combination therapy did not induce significant changes in body weight, blood composition, plasma creatinine levels and renal morphology, indicating the absence of severe acute hematologic and renal toxicity. Conclusion: These results highlight that the potentiation of the therapeutic effect of PRRT by DNA-PKcs inhibition is a highly effective and well-tolerated therapeutic strategy. Based on our findings, we recommend initiation of phase I/II studies in patients to find a safe and effective combination regimen.

Radiolabelling of Polyclonally Expanded Human Regulatory T Cells (Treg) with 89Zr-oxine for Medium-Term In Vivo Cell Tracking.

Regulatory T cells (Tregs) are a promising candidate cell therapy to treat autoimmune diseases and aid the longevity of transplanted solid organs. Despite increasing numbers of clinical trials using human Treg therapy, important questions pertaining to their in vivo fate, distribution, and function remain unanswered. Treg accumulation in relevant tissues was found to be crucial for Treg therapy efficacy, but existing blood-borne biomarkers are unlikely to accurately reflect the tissue state. Non-invasive Treg tracking by whole-body imaging is a promising alternative and can be achieved by direct radiolabelling of Tregs and following the radiolabelled cells with positron emission tomography (PET). Our goal was to evaluate the radiolabelling of polyclonal Tregs with 89Zr to permit their in vivo tracking by PET/CT for longer than one week with current preclinical PET instrumentation. We used [89Zr]Zr(oxinate)4 as the cell-labelling agent and achieved successful radiolabelling efficiency of human Tregs spanning 0.1-11.1 Bq 89Zr/Treg cell, which would be compatible with PET tracking beyond one week. We characterized the 89Zr-Tregs, assessing their phenotypes, and found that they were not tolerating these intracellular 89Zr amounts, as they failed to survive or expand in a 89Zr-dose-dependent manner. Even at 0.1 Bq 89Zr per Treg cell, while 89Zr-Tregs remained functional as determined by a five-day-long effector T cell suppression assay, they failed to expand beyond day 3 in vitro. Moreover, PET imaging revealed signs of 89Zr-Treg death after adoptive transfer in vivo. In summary, 89Zr labelling of Tregs at intracellular radioisotope amounts compatible with cell tracking over several weeks did not achieve the desired outcomes, as 89Zr-Tregs failed to expand and survive. Consequently, we conclude that indirect Treg labelling is likely to be the most effective alternative method to satisfy the requirements of this cell tracking scenario.

Epigenetic drugs in somatostatin type 2 receptor radionuclide theranostics and radiation transcriptomics in mouse pheochromocytoma models.

Pheochromocytomas and paragangliomas (PCCs/PGLs) are catecholamine-producing tumors. In inoperable and metastatic cases, somatostatin type 2 receptor (SSTR2) expression allows for peptide receptor radionuclide therapy with [177Lu]Lu-DOTA-TATE. Insufficient receptor levels, however, limit treatment efficacy. This study evaluates whether the epigenetic drugs valproic acid (VPA) and 5-Aza-2'-deoxycytidine (DAC) modulate SSTR2 levels and sensitivity to [177Lu]Lu-DOTA-TATE in two mouse PCC models (MPC and MTT). Methods: Drug-effects on Sstr2/SSTR2 were investigated in terms of promoter methylation, mRNA and protein levels, and radiotracer binding. Radiotracer uptake was measured in subcutaneous allografts in mice using PET and SPECT imaging. Tumor growth and gene expression (RNAseq) were characterized after drug treatments. Results: DAC alone and in combination with VPA increased SSTR2 levels along with radiotracer uptake in vitro in MPC (high-SSTR2) and MTT cells (low-SSTR2). MTT but not MPC allografts responded to DAC and VPA combination with significantly elevated radiotracer uptake, although activity concentrations remained far below those in MPC tumors. In both models, combination of DAC, VPA and [177Lu]Lu-DOTA-TATE was associated with additive effects on tumor growth delay and specific transcriptional responses in gene sets involved in cancer and treatment resistance. Effects of epigenetic drugs were unrelated to CpG island methylation of the Sstr2 promoter. Conclusion: This study demonstrates that SSTR2 induction in mouse pheochromocytoma models has some therapeutic benefit that occurs via yet unknown mechanisms. Transcriptional changes in tumor allografts associated with epigenetic treatment and [177Lu]Lu-DOTA-TATE provide first insights into genetic responses of PCCs/PGLs, potentially useful for developing additional strategies to prevent tumor recurrence.

Novel NK1R-Targeted 68Ga-/177Lu-Radioconjugates with Potential Application against Glioblastoma Multiforme: Preliminary Exploration of Structure-Activity Relationships.

Locoregionally administered, NK1 receptor (NK1R) targeted radionuclide therapy is a promising strategy for the treatment of glioblastoma multiforme. So far, the radiopharmaceuticals used in this approach have been based on the endogenous agonist of NK1R, Substance P or on its close analogues. Herein, we used a well-known, small molecular NK1R antagonist, L732,138, as the basis for the radiopharmaceutical vector. First, 14 analogues of this compound were evaluated to check whether extending the parent structure with linkers of different lengths would not deteriorate the NK1R binding. The tested analogues had affinity similar to or better than the parent compound, and none of the linkers had a negative impact on the binding. Next, five DOTA conjugates were synthesized and used for labelling with 68Ga and 177Lu. The obtained radioconjugates turned out to be fairly lipophilic but showed rather limited stability in human plasma. Evaluation of the receptor affinity of the (radio)conjugates showed that neither the chelator nor the metal negatively impacts the NK1R binding. The 177Lu-radioconjugates exhibited the binding characteristics towards NK1R similar or better than that of the 177Lu-labelled derivative of Substance P, which is in current clinical use. The experimental results presented herein, along with their structural rationalization provided by modelling, give insight for the further molecular design of small molecular NK1R-targeting vectors.

Translational Development of a Zr-89-Labeled Inhibitor of Prostate-specific Membrane Antigen for PET Imaging in Prostate Cancer.

PURPOSE: We present here a Zr-89-labeled inhibitor of prostate-specific membrane antigen (PSMA) as a complement to the already established F-18- or Ga-68-ligands. PROCEDURES: The precursor PSMA-DFO (ABX) was used for Zr-89-labeling. This is not an antibody, but a peptide analogue of the precursor for the production of [177Lu]Lu-PSMA-617. The ligand [89Zr]Zr-PSMA-DFO was compared with [68Ga]Ga-PSMA-11 and [18F]F-JK-PSMA-7 in vitro by determination of the Kd value, cellular uptake, internalization in LNCaP cells, biodistribution studies with LNCaP prostate tumor xenografts in mice, and in vivo by small-animal PET imaging in LNCaP tumor mouse models. A first-in-human PET was performed with [89Zr]Zr-PSMA-DFO on a patient presenting with a biochemical recurrence after brachytherapy and an ambiguous intraprostatic finding with [18F]F-JK-PSMA-7 but histologically benign cells in a prostate biopsy 7 months previously. RESULTS: [89Zr]Zr-PSMA-DFO was prepared with a radiochemical purity ≥ 99.9% and a very high in vitro stability for up to 7 days at 37 °C. All radiotracers showed similar specific cellular binding and internalization, in vitro and comparable tumor uptake in biodistribution experiments during the first 5 h. The [89Zr]Zr-PSMA-DFO achieved significantly higher tumor/background ratios in LNCaP tumor xenografts (tumor/blood: 309 ± 89, tumor/muscle: 450 ± 38) after 24 h than [68Ga]Ga-PSMA-11 (tumor/blood: 112 ± 57, tumor/muscle: 58 ± 36) or [18F]F-JK-PSMA-7 (tumor/blood: 175 ± 30, tumor/muscle: 114 ± 14) after 4 h (p < 0.01). Small-animal PET imaging demonstrated in vivo that tumor visualization with [89Zr]Zr-PSMA-DFO is comparable to [68Ga]Ga-PSMA-11 or [18F]F-JK-PSMA-7 at early time points (1 h p.i.) and that PET scans up to 48 h p.i. clearly visualized the tumor at late time points. A late [89Zr]Zr-PSMA-DFO PET scan on a patient with biochemical recurrence (BCR) had demonstrated intensive tracer accumulation in the right (SUVmax 13.25, 48 h p.i.) and in the left prostate lobe (SUV max 9.47), a repeat biopsy revealed cancer cells on both sides. CONCLUSION: [89Zr]Zr-PSMA-DFO is a promising PSMA PET tracer for detection of tumor areas with lower PSMA expression and thus warrants further clinical evaluation.

Improving Theranostic Gallium-68/Lutetium-177-Labeled PSMA Inhibitors with an Albumin Binder for Prostate Cancer.

We developed a novel therapeutic radioligand, [177Lu]1h, with an albumin binding motif and evaluated it in a prostate-specific membrane antigen (PSMA)-expressing tumor xenograft mouse model. Fourteen PSMA target candidates were synthesized, and binding affinity was evaluated with an in vitro competitive binding assay. First, four compound candidates were selected depending on binding affinity results. Next, we selected four compounds ([68Ga]1e, [68Ga]1g, [68Ga]1h, and [68Ga]1k) were screened for tumor targeting efficiency by micro-positron emission tomography/computed tomography (micro-PET/CT) imaging. Finally, [177Lu]1h compound was evaluated the tumor targeting efficiency and therapeutic efficiency by micro-single-photon emission computed tomography/computed tomography (micro-SPECT/CT), biodistribution, and radiotherapy studies. Estimated human effective dose was calculated by biodistribution data. Compound 1h showed a high binding affinity (Ki value = 4.08 ± 0.08 nmol/L), and [177Lu]1h showed extended blood circulation (1 hour = 10.32 ± 0.31, 6 hours = 2.68 ± 1.07%ID/g) compared to [177Lu]PSMA-617 (1 h = 0.17 ± 0.10%ID/g). [177Lu]1h was excreted via the renal pathway and showed high tumor uptake (24.43 ± 3.36%ID/g) after 1 hour, which increased over 72 hours (72 hours = 51.39 ± 9.26%ID/g). Mice treated with 4 and 6 MBq of [177Lu]1h showed a median survival rate of >61 days. In particular, all mice treated with 6 MBq of [177Lu]1h survived for the entire monitoring period. The estimated human effective dose of [177Lu]1h was 0.07 ± 0.01 and 0.03 ± 0.00 mSv/MBq in total body and kidney, respectively. The current study indicates that [177Lu]1h has the potential for further investigation of metastatic castration-resistant prostate cancer (mCRPC) therapy in clinical trials.

Spatio-temporal biodistribution of 89Zr-oxine labeled huLym-1-A-BB3z-CAR T-cells by PET imaging in a preclinical tumor model.

Quantitative in vivo monitoring of cell biodistribution offers assessment of treatment efficacy in real-time and can provide guidance for further optimization of chimeric antigen receptor (CAR) modified cell therapy. We evaluated the utility of a non-invasive, serial 89Zr-oxine PET imaging to assess optimal dosing for huLym-1-A-BB3z-CAR T-cell directed to Lym-1-positive Raji lymphoma xenograft in NOD Scid-IL2Rgammanull (NSG) mice. In vitro experiments showed no detrimental effects in cell health and function following 89Zr-oxine labeling. In vivo experiments employed simultaneous PET/MRI of Raji-bearing NSG mice on day 0 (3 h), 1, 2, and 5 after intravenous administration of low (1.87 ± 0.04 × 106 cells), middle (7.14 ± 0.45 × 106 cells), or high (16.83 ± 0.41 × 106 cells) cell dose. Biodistribution (%ID/g) in regions of interests defined over T1-weighted MRI, such as blood, bone, brain, liver, lungs, spleen, and tumor, were analyzed from PET images. Escalating doses of CAR T-cells resulted in dose-dependent %ID/g biodistributions in all regions. Middle and High dose groups showed significantly higher tumor %ID/g compared to Low dose group on day 2. Tumor-to-blood ratios showed the enhanced extravascular tumor uptake by day 2 in the Low dose group, while the Middle dose showed significant tumor accumulation starting on day 1 up to day 5. From these data obtained over time, it is apparent that intravenously administered CAR T-cells become trapped in the lung for 3-5 h and then migrate to the liver and spleen for up to 2-3 days. This surprising biodistribution data may be responsible for the inactivation of these cells before targeting solid tumors. Ex vivo biodistributions confirmed in vivo PET-derived biodistributions. According to these studies, we conclude that in vivo serial PET imaging with 89Zr-oxine labeled CAR T-cells provides real-time monitoring of biodistributions crucial for interpreting efficacy and guiding treatment in patient care.

In Vitro Evaluation of No-Carrier-Added Radiolabeled Cisplatin ([189, 191Pt]cisplatin) Emitting Auger Electrons.

Due to their short-range (2-500 nm), Auger electrons (Auger e-) have the potential to induce nano-scale physiochemical damage to biomolecules. Although DNA is the primary target of Auger e-, it remains challenging to maximize the interaction between Auger e- and DNA. To assess the DNA-damaging effect of Auger e- released as close as possible to DNA without chemical damage, we radio-synthesized no-carrier-added (n.c.a.) [189, 191Pt]cisplatin and evaluated both its in vitro properties and DNA-damaging effect. Cellular uptake, intracellular distribution, and DNA binding were investigated, and DNA double-strand breaks (DSBs) were evaluated by immunofluorescence staining of γH2AX and gel electrophoresis of plasmid DNA. Approximately 20% of intracellular radio-Pt was in a nucleus, and about 2% of intra-nucleus radio-Pt bound to DNA, although uptake of n.c.a. radio-cisplatin was low (0.6% incubated dose after 25-h incubation), resulting in the frequency of cells with γH2AX foci was low (1%). Nevertheless, some cells treated with radio-cisplatin had γH2AX aggregates unlike non-radioactive cisplatin. These findings suggest n.c.a. radio-cisplatin binding to DNA causes severe DSBs by the release of Auger e- very close to DNA without chemical damage by carriers. Efficient radio-drug delivery to DNA is necessary for successful clinical application of Auger e-.

The chemical tool-kit for molecular imaging with radionuclides in the age of targeted and immune therapy.

Nuclear medicine has evolved over the last half-century from a functional imaging modality using a handful of radiopharmaceuticals, many of unknown structure and mechanism of action, into a modern speciality that can properly be described as molecular imaging, with a very large number of specific radioactive probes of known structure that image specific molecular processes. The advances of cancer treatment in recent decades towards targeted and immune therapies, combined with recognition of heterogeneity of cancer cell phenotype among patients, within patients and even within tumours, has created a growing need for personalised molecular imaging to support treatment decision. This article describes the evolution of the present vast range of radioactive probes - radiopharmaceuticals - leveraging a wide variety of chemical disciplines, over the last half century. These radiochemical innovations have been inspired by the need to support personalised medicine and also by the parallel development in development of new radionuclide imaging technologies - from gamma scintigraphy, through single photon emission tomography (SPECT), through the rise of clinical positron emission tomography (PET) and PET-CT, and perhaps in the future, by the advent of total body PET. Thus, in the interdisciplinary world of nuclear medicine and molecular imaging, as quickly as radiochemistry solutions are developed to meet new needs in cancer imaging, new challenges emerge as developments in one contributing technology drive innovations in the others.

Imaging atherosclerotic plaques by targeting Galectin-3 and activated macrophages using (89Zr)-DFO- Galectin3-F(ab')2 mAb.

Rationale: The high expression of Galectin-3 (Gal3) in macrophages of atherosclerotic plaques suggests its participation in atherosclerosis pathogenesis, and raises the possibility to use it as a target to image disease severity in vivo. Here, we explored the feasibility of tracking atherosclerosis by targeting Gal3 expression in plaques of apolipoprotein E knockout (ApoE-KO) mice via PET imaging. Methods: Targeting of Gal3 in M0-, M1- and M2 (M2a/M2c)-polarized macrophages was assessed in vitro using a Gal3-F(ab')2 mAb labeled with AlexaFluor®488 and 89Zr- desferrioxamine-thioureyl-phenyl-isothiocyanate (DFO). To visualize plaques in vivo, ApoE-KO mice were injected i.v. with 89Zr-DFO-Gal3-F(ab')2 mAb and imaged via PET/CT 48 h post injection. Whole length aortas harvested from euthanized mice were processed for Sudan-IV staining, autoradiography, and immunostaining for Gal3, CD68 and α-SMA expression. To confirm accumulation of the tracer in plaques, ApoE-KO mice were injected i.v. with Cy5.5-Gal3-F(ab')2 mAb, euthanized 48 h post injection, followed by cryosections of the body and acquisition of fluorescent images. To explore the clinical potential of this imaging modality, immunostaining for Gal3, CD68 and α-SMA expression were carried out in human plaques. Single cell RNA sequencing (scRNA-Seq) analyses were performed to measure LGALS3 (i.e. a synonym for Gal3) gene expression in each macrophage of several subtypes present in murine or human plaques. Results: Preferential binding to M2 macrophages was observed with both AlexaFluor®488-Gal3-F(ab')2 and 89Zr-DFO-Gal3-F(ab')2 mAbs. Focal and specific 89Zr-DFO-Gal3-F(ab')2 mAb uptake was detected in plaques of ApoE-KO mice by PET/CT. Autoradiography and immunohistochemical analyses of aortas confirmed the expression of Gal3 within plaques mainly in macrophages. Moreover, a specific fluorescent signal was visualized within the lesions of vascular structures burdened by plaques in mice. Gal3 expression in human plaques showed similar Gal3 expression patterns when compared to their murine counterparts. Conclusions: Our data reveal that 89Zr-DFO-Gal3-F(ab')2 mAb PET/CT is a potentially novel tool to image atherosclerotic plaques at different stages of development, allowing knowledge-based tailored individual intervention in clinically significant disease.

An in silico study on the effect of the radionuclide half-life on PET/CT imaging with PSMA-targeting radioligands.

AIM: The aim of this work was to systematically investigate the influence of the radionuclide half-life and affinity of prostate-specific membrane antigen (PSMA)-targeting ligands on the activity concentration for PET/CT imaging. METHODS: A whole-body physiologically-based pharmacokinetic (PBPK) model with individually estimated parameters of 13 patients with metastatic castration-resistant prostate cancer (mCRPC) was used to simulate the pharmacokinetics of PSMA-targeting radioligands. The simulations were performed with 68Ga (T1/2 = 1.13 h), 18F (T1/2 = 1.83 h), 64Cu (T1/2 = 12.7 h) and for different affinities (dissociation constants KD of 1-0.01 nM) and a commonly used ligand amount of 3 nmol. The activity concentrations were calculated at 1, 2, 3, 4, 8, 12, and 16 h after injection. RESULTS: The highest tumor uptake was achieved 1 h p. i. for 68Ga-PSMA. For 18F-PSMA, the highest tumor uptake was at 1 h p. i. and 2 h p.i for dissociation constants KD  = 1 nM and KD  = 0.1-0.01 nM, respectively. For 64Cu-PSMA, the highest tumor uptake was at 4 h p. i. for dissociation constant KD  = 1 nM and at 4 h p. i. (9 patients) and 8 h p. i. (4 patients) for higher affinities. Compared to 68Ga-PSMA (1 h p. i.), the activity concentrations in the tumor for 18F-PSMA (2 h p. i.) increased maximum 1.3-fold with minor differences for all affinities. For 64Cu-PSMA (4 h p. i.), the improvements were in the range of 2.8 to 3.2-fold for all affinities. CONCLUSIONS: The simulations indicate that the highest tumor-to-background ratio can be achieved after 4 hours in PET/CT using high-affinity 64Cu-PSMA.

Initial In Vitro and In Vivo Evaluation of a Novel CCK2R Targeting Peptide Analog Labeled with Lutetium-177.

Targeting of cholecystokinin-2 receptor (CCK2R) expressing tumors using radiolabeled minigastrin (MG) analogs is hampered by rapid digestion of the linear peptide in vivo. In this study, a new MG analog stabilized against enzymatic degradation was investigated in preclinical studies to characterize the metabolites formed in vivo. The new MG analog DOTA-DGlu-Pro-Tyr-Gly-Trp-(N-Me)Nle-Asp-1Nal-NH2 comprising site-specific amino acid substitutions in position 2, 6 and 8 and different possible metabolites thereof were synthesized. The receptor interaction of the peptide and selected metabolites was evaluated in a CCK2R-expressing cell line. The enzymatic stability of the 177Lu-labeled peptide analog was evaluated in vitro in different media as well as in BALB/c mice up to 1 h after injection and the metabolites were identified based on radio-HPLC analysis. The new radiopeptide showed a highly increased stability in vivo with >56% intact radiopeptide in the blood of BALB/c mice 1 h after injection. High CCK2R affinity and cell uptake was confirmed only for the intact peptide, whereas enzymatic cleavage within the receptor specific C-terminal amino acid sequence resulted in complete loss of affinity and cell uptake. A favorable biodistribution profile was observed in BALB/c mice with low background activity, preferential renal excretion and prolonged uptake in CCK2R-expressing tissues. The novel stabilized MG analog shows high potential for diagnostic and therapeutic use. The radiometabolites characterized give new insights into the enzymatic degradation in vivo.

188Re-labeled GX1 dimer as a novel dual-functional probe targeting TGM2 for imaging and antiangiogenic therapy of gastric cancer.

PURPOSE: The GX1 peptide (CGNSNPKSC) can specifically bind to TGM2 and possesses the ability to target the blood vessels of gastric cancer. This study intends to develop an integrated dual-functional probe with higher affinity, specificity and targeting and to characterize it in vivo and in vitro. METHODS: The dimer and tetramer of GX1 were prepared using cross-linked PEG and labeled with 99mTc. The best targeting probe [PEG-(GX1)2] was selected by gamma camera imaging in nude mouse models of gastric cancer. 188Re-PEG-(GX1)2 was prepared and characterized through cell binding analysis and competitive inhibition experiments, gamma camera imaging, MTT analysis and flow cytometry, BLI, immunohistochemistry, HE staining and biochemical analysis. RESULTS: PEG-(GX1)2 bound specifically to Co-HUVEC with higher affinity than GX1. 188Re-PEG-(GX1)2 had better ability to target gastric cancer in tumor-bearing nude mice and higher T/H ratios than 188Re-GX1. 188Re-PEG-(GX1)2 inhibited the growth of Co-HUVEC and induced apoptosis, and its effects were more robust than those of 188Re-GX1. BLI showed that 188Re-PEG-(GX1)2 inhibited tumor proliferation in vivo with a stronger effect than 188Re-GX1. Compared with 188Re-GX1, 188Re-PEG-(GX1)2 suppressed tumor angiogenesis and tumor cell proliferation and induced tumor cell apoptosis in vivo. The 188Re-PEG-(GX1)2 group did not cause visible changes in liver and kidney morphology and function in vivo. CONCLUSION: The dimer of GX1 was synthesized by using cross-linked PEG, and then 188Re-PEG-(GX1)2 was prepared. This radiopharmaceutical played both diagnostic and therapeutic functions, and gamma camera imaging could be utilized to detect the distribution of drugs in vivo during treatment. Through a series of experiments in vitro and in vivo, the feasibility of the drug was confirmed, and these results laid the foundation for the subsequent development and application of GX1.

Studies of selenium and arsenic mutual protection in human HepG2 cells.

Hundreds of millions of people worldwide are exposed to unacceptable levels of carcinogenic inorganic arsenic. Animal models have shown that selenium and arsenic are mutually protective through the formation and elimination of the seleno-bis(S-glutathionyl) arsinium ion [(GS)2AsSe]-. Consistent with this, human selenium deficiency in arsenic-endemic regions is associated with arsenic-induced disease, leading to the initiation of human selenium supplementation trials. In contrast to the protective effect observed in vivo, in vitro studies have suggested that selenite increases arsenite cellular retention and toxicity. This difference might be explained by the rapid conversion of selenite to selenide in vivo. In the current study, selenite did not protect the human hepatoma (HepG2) cell line against the toxicity of arsenite at equimolar concentrations, however selenide increased the IC50 by 2.3-fold. Cytotoxicity assays of arsenite + selenite and arsenite + selenide at different molar ratios revealed higher overall mutual antagonism of arsenite + selenide toxicity than arsenite + selenite. Despite this protective effect, in comparison to 75Se-selenite, HepG2 cells in suspension were at least 3-fold more efficient at accumulating selenium from reduced 75Se-selenide, and its accumulation was further increased by arsenite. X-ray fluorescence imaging of HepG2 cells also showed that arsenic accumulation, in the presence of selenide, was higher than in the presence of selenite. These results are consistent with a greater intracellular availability of selenide relative to selenite for protection against arsenite, and the formation and retention of a less toxic product, possibly [(GS)2AsSe]-.

Molecular imaging in lymphoma beyond 18F-FDG-PET: understanding the biology and its implications for diagnostics and therapy.

Mature lymphoproliferative diseases are a heterogeneous group of neoplasms arising from different stages of B-cell and T-cell development. With improved understanding of the molecular processes in lymphoma and novel treatment options, arises a growing need for the molecular characterisation of tumours. Molecular imaging with single-photon-emission CT and PET using specific radionuclide tracers can provide whole-body information to investigate cancer biology, to evaluate phenotypic heterogeneity, to identify resistance to targeted therapy, and to assess the biodistribution of drugs in patients. In this Review, we evaluate the existing literature on molecular imaging in lymphoma, other than 18F-fluordeoxyglucose molecular imaging. The aim is to examine the contribution of molecular imaging to the understanding of the biology of lymphoma and to discuss potential implications for the diagnostics and therapy of this disease. Finally, we discuss possible applications for molecular imaging of patients with lymphoma in the clinical context.

Cetuximab PET delineated changes in cellular distribution of EGFR upon dasatinib treatment in triple negative breast cancer.

BACKGROUND: At least 50% of triple negative breast cancer (TNBC) overexpress the epidermal growth factor receptor, EGFR, which paved the way for clinical trials investigating its blockade. Outcomes remained dismal stemming from mechanisms of resistance particularly the nuclear cycling of EGFR, which is enhanced by Src activation. Attenuation of Src reversed nuclear translocation, restoring EGFR to the cell surface. Herein, we hypothesize that changes in cellular distribution of EGFR upon Src inhibition with dasatinib can be annotated through the EGFR immunopositron emission tomography (immunoPET) radiotracer, [89Zr]Zr-cetuximab. METHODS: Nuclear and non-nuclear EGFR levels of dasatinib-treated vs. untreated MDA-MB-231 and MDA-MB-468 cells were analyzed via immunoblots. Both treated and untreated cells were exposed to [89Zr]Zr-cetuximab to assess binding at 4 °C and 37 °C. EGFR-positive MDA-MB-231, MDA-MB-468, and a patient-derived xenograft were treated with dasatinib or vehicle followed by cetuximab PET imaging to compare EGFR levels. After imaging, the treated mice were separated into two groups: one cohort continued with dasatinib with the addition of cetuximab while the other cohort received dasatinib alone. Correlations between the radiotracer uptake vs. changes in tumor growth and EGFR expression from immunoblots were analyzed. RESULTS: Treated cells displayed higher binding of [89Zr]Zr-cetuximab to the cell membrane at 4 °C and with greater internalized activity at 37 °C vs. untreated cells. In all tumor models, higher accumulation of the radiotracer in dasatinib-treated groups was observed compared to untreated tumors. Treated tumors displayed significantly decreased pSrc (Y416) with retained total Src levels compared to control. In MDA-MB-468 and PDX tumors, the analysis of cetuximab PET vs. changes in tumor volume showed an inverse relationship where high tracer uptake in the tumor demonstrated minimal tumor volume progression. Furthermore, combined cetuximab and dasatinib treatment showed better tumor regression compared to control and dasatinib-only-treated groups. No benefit was achieved in MDA-MB-231 xenografts with the addition of cetuximab, likely due to its KRAS-mutated status. CONCLUSIONS: Cetuximab PET can monitor effects of dasatinib on EGFR cellular distribution and potentially inform treatment response in wild-type KRAS TNBC.