One of the challenges in optimizing PSMA-based therapies for the treatment of prostate cancer is the stability of the radiopharmaceutical, defined by the degradation of radiolabeled compounds due to exposure to ionizing radiation (radiolysis). Radiolysis of a radiopharmaceutical is difficult to measure and can influence the biodistrubion of radiopharmaceuticals in vivo, possibly leading to toxicity or suboptimal outcomes. Therefore, an inter-laboratory study was performed to confirm accurate detection of radiolysed [177Lu]Lu-PSMA-I T, and the biodistribution of radiolysed radiopharmaceutical was investigated in vivo, and HPLC methods are compared. Multiple HPLC-methods were compared to evaluate the analysis of degraded radiopharmaceutical and an inter-laboratory comparison was conducted to evaluate the consistency of current analytical methods across various institutions and with current methods from literature. Additionally, for the biodistribution experiments, a radiolabeling of [177Lu]Lu-PSMA-I T was adjusted to achieve a radiochemical purity of 50

The cholecystokinin type 2 receptor (CCK2R), which mediates the effects of gastrin and cholecystokinin (CCK), is known to promote colorectal cancer (CRC) proliferation in advanced stages. Though well-studied in gastric and pancreatic cancers, the role of CCK2R in colorectal, especially rectal cancer (RC), is still emerging. In this research, we initially evaluated CCK2R expression across rectal and colorectal cancer cell lines using qRT-PCR to establish baseline expression patterns. To explore the potential for near-infrared (NIR) fluorescence-based tumor targeting, we synthesized a CCK2R-binding peptide conjugate, Cy5@PP-F11, and demonstrated its receptor-specific uptake in vitro through live-cell imaging microscopy. While these results suggest promise for tumor targeting, further studies are needed to assess its in vivo performance and utility in fluorescence-guided surgery. In addition, a cohort of 495 Stage I-IV rectal cancer patients was analyzed using tissue microarrays (TMAs) and immunohistochemistry (IHC) to assess CCK2R expression in both tumor and normal tissues. Expression patterns and their prognostic significance were evaluated in relation to clinicopathological parameters. CCK2R localization and expression levels were quantified in both the epithelial and stromal tumor compartments, with cytoplasmic and nuclear staining specifically analyzed in the epithelial compartment. Scoring data were assessed to determine their clinical relevance. Our findings revealed significant overexpression of CCK2R in both epithelial and stromal compartments of rectal cancer tissues compared to normal tissues, showing its high suitability as target for tumor imaging. While epithelial CCK2R expression showed no significant link to clinical staging, stromal expression was notably involved, indicating a role in tumor-stroma interactions and the tumor microenvironment.

Radiolabelled minigastrin (MG) analogues targeting the cholecystokinin-2 receptor (CCK2R) have proven to be a promising approach for peptide receptor radionuclide therapy (PRRT). In this study, we report on the radiopharmaceutical development and standardization of the preparation of [177Lu]Lu-DOTA-MGS5 using an automated synthesis module. Furthermore, we present the preclinical tests required to move forward towards a first therapeutic clinical trial as well as preliminary clinical dosimetry data. Five individual batches of [177Lu]Lu-DOTA-MGS5 were synthesized and analysed according to predefined quality control specifications. Cell-based experiments and biodistribution studies were performed to evaluate the specific receptor binding and tumour uptake of the radiopharmaceutical formulation. A preclinical dosimetry study was carried out in tumour xenografted mice and a first dosimetry study was performed in a patient with small cell lung cancer. The automated cassette-based production of [177Lu]Lu-DOTA-MGS5 resulted in a product with high radiochemical purity of > 98

Incomplete resection of prostate cancer (PCa) results in increased risk of disease recurrence. Combined fluorescence-guided surgery with tumor-targeted photodynamic therapy (tPDT) may help to achieve complete tumor eradication. We developed a prostate-specific membrane antigen (PSMA) ligand consisting of a DOTA chelator for 111In labeling and a fluorophore/photosensitizer IRDye700DX (PSMA-N064). We evaluated the efficacy of PSMA-tPDT using PSMA-N064 in cell viability assays, a mouse xenograft model and in an ex vivo incubation study on fresh human PCa tissue.In vitro, therapeutic efficacy of PSMA-N064 was evaluated using PSMA-positive LS174T cells and LS174T wild-type cells. In vivo, PSMA-N064-mediated tPDT was tested in immunodeficient BALB/c mice-bearing PSMA-positive LS174T xenografts. Tumor growth and survival were compared to control mice that received either NIR light or ligand injection only. Ex vivo tPDT efficacy was evaluated in excised fresh human PCa tissue incubated with PSMA-N064.In vitro, tPDT led to a PSMA-specific light- and ligand dose-dependent loss in cell viability. In vivo, tPDT-induced tumor cell apoptosis, delayed tumor growth, and significantly improved survival (p = 0.004) of the treated PSMA-positive tumor-bearing mice compared with the controls. In fresh ex vivo human PCa tissue, apoptosis was significantly increased in PSMA-tPDT-treated samples compared to non-treated control samples (p = 0.037).This study showed the feasibility of PSMA-N064-mediated tPDT in cell assays, a xenograft model and excised fresh human PCa tissue. This paves the way to investigate the impact of in vivo PSMA-tPDT on surgical outcome in PCa patients.

Polyisocyanopeptide (PIC) hydrogels are proposed as promising wound dressings. These gels are thermo-sensitive, allow application as a cold liquid, and rely on gelation through body heat. It is supposed that the gel can be easily removed by reversing the gelation and washing it away with a cold irrigation solution. The impact on wound healing of the regular application and removal of PIC dressings is compared to a single application of PIC and the clinically used Tegaderm™ in murine splinted full-thickness wounds for up to 14 days. SPECT/CT analysis of 111In-labelled PIC gels showed that, on average, 58% of the PIC gel could be washed out of the wounds with the employed method, which is, however, heavily influenced by personal technique. Evaluation with photography and (immuno-)histology showed that wounds in which PIC dressings were regularly removed and replaced were smaller at 14 days post-injury but performed on par with the control treatment. Moreover, the encapsulation of PIC in wound tissue was less severe and occurred less often when PIC was regularly refreshed. In addition, no morphological damage related to the removal procedure was observed. Thus, PIC gels are atraumatic and perform similarly to currently employed wound dressing materials, offering possible future benefits for both clinicians and patients.

Imaging with radiolabeled exendin enables detection and characterization of glucagon-like peptide 1 receptors (GLP-1Rs) in vivo with high specificity. The novel radiotracer [68Ga]Ga-NODAGA-exendin-4 forms a stable complex after a simple and fast labeling procedure. Beta-cell mass in the islets of Langerhans can be visualized using [68Ga]Ga-NODAGA-exendin-4, which is promising for research into diabetes mellitus (DM) pathophysiology. Furthermore, this radiotracer enables very sensitive detection of insulinomas, resulting from vast overexpression of GLP-1Rs, and seems promising for the detection of focal lesions in congenital hyperinsulinism (CHI). Here, we describe the procedures involved in [68Ga]Ga-NODAGA-exendin-4 positron emission tomography (PET)/computed tomography (CT) imaging including the radiolabeling of the NODAGA-exendin conjugate with 68Ga, quality controls, and PET/CT.

The therapeutic potential of minigastrin (MG) analogs for the treatment of cholecystokinin-2 receptor (CCK2R)-expressing cancers is limited by poor in vivo stability or unfavorable accumulation in non-target tissues. Increased stability against metabolic degradation was achieved by modifying the C-terminal receptor-specific region. This modification led to significantly improved tumor targeting properties. In this study, further N-terminal peptide modifications were investigated. Two novel MG analogs were designed starting from the amino acid sequence of DOTA-MGS5 (DOTA-DGlu-Ala-Tyr-Gly-Trp-(N-Me)Nle-Asp-1Nal-NH2). Introduction of a penta-DGlu moiety and replacement of the four N-terminal amino acids by a non-charged hydrophilic linker was investigated. Retained receptor binding was confirmed using two CCK2R-expressing cell lines. The effect on metabolic degradation of the new 177Lu-labeled peptides was studied in human serum in vitro, as well as in BALB/c mice in vivo. The tumor targeting properties of the radiolabeled peptides were assessed using BALB/c nude mice bearing receptor-positive and receptor-negative tumor xenografts. Both novel MG analogs were found to have strong receptor binding, enhanced stability, and high tumor uptake. Replacement of the four N-terminal amino acids by a non-charged hydrophilic linker lowered the absorption in the dose-limiting organs, whereas introduction of the penta-DGlu moiety increased uptake in renal tissue.

Abstract Objectives OA is characterized by cartilage degeneration and persistent pain. The majority of OA patients present with synovitis, which is associated with increased cartilage damage. Activated synovial macrophages are key contributors to joint destruction. Therefore, a marker that reflects the activation of these cells could be a valuable tool to characterize the destructive potential of synovitis and benefit monitoring of OA. Here, we aimed to investigate the use of CD64 (FcγRI) as a marker to characterize the damaging potential of synovitis in OA. Methods Synovial biopsies were obtained from end-stage OA patients that underwent joint replacement surgery. CD64 protein expression and localization was evaluated using immunohistochemistry and immunofluorescence and quantified using flow cytometry. qPCR was performed to measure the expression of FCGR1 and OA-related genes in synovial biopsies, and in primary chondrocytes and primary fibroblasts stimulated with OA conditioned medium (OAS-CM). Results Our data exposed a wide range of CD64 expression in OA synovium and showed positive correlations between FCGR1 and S100A8, S100A9, IL1B, IL6 and MMP1/2/3/9/13 expression. CD64 protein correlated with MMP1, MMP3, MMP9, MMP13 and S100A9. Furthermore, we observed that synovial CD64 protein levels in source tissue for OAS-CM significantly associated with the OAS-CM-induced expression of MMP1, MMP3 and especially ADAMTS4 in cultured fibroblasts, but not chondrocytes. Conclusion Together, these results indicate that synovial CD64 expression is associated with the expression of proteolytic enzymes and inflammatory markers related to structural damage in OA. CD64 therefore holds promise as marker to characterize the damaging potential of synovitis.

Abstract Objective Activated synovial fibroblasts are key effector cells in RA. Selectively depleting these based upon their expression of fibroblast activation protein (FAP) is an attractive therapeutic approach. Here we introduce FAP imaging of inflamed joints using 68Ga-FAPI-04 in a RA patient, and aim to assess feasibility of anti-FAP targeted photodynamic therapy (FAP-tPDT) ex vivo using 28H1-IRDye700DX on RA synovial explants. Methods Remnant synovial tissue from RA patients was processed into 6 mm biopsies and, from several patients, into primary fibroblast cell cultures. Both were treated using FAP-tPDT. Cell viability was measured in fibroblast cultures and biopsies were evaluated for histological markers of cell damage. Selectivity of the effect of FAP-tPDT was assessed using flow cytometry on primary fibroblasts and co-cultured macrophages. Additionally, one RA patient intravenously received 68Ga-FAPI-04 and was scanned using PET/CT imaging. Results In the RA patient, FAPI-04 PET imaging showed high accumulation of the tracer in arthritic joints with very low background signal. In vitro, FAP-tPDT induced cell death in primary RA synovial fibroblasts in a light dose-dependent manner. An upregulation of cell damage markers was observed in the synovial biopsies after FAP-tPDT. No significant effects of FAP-tPDT were noted on macrophages after FAP-tPDT of neighbouring fibroblasts. Conclusion In this study the feasibility of selective FAP-tPDT in synovium of rheumatoid arthritis patients ex vivo is demonstrated. Furthermore, this study provides the first indication that FAP-targeted PET/CT can be used to image arthritic joints, an important step towards application of FAP-tPDT as a targeted locoregional therapy for RA.

Minigastrin (MG) analogs for therapy of CCK2R-expressing malignancies are limited by low stability in vivo or excessive accumulation in non-target organs. By modifying the C-terminal receptor-binding sequence, metabolization could be prevented and tumor targeting significantly improved. In this work, N-terminal changes of the peptide length were evaluated. Based on the amino acid sequence of DOTA-MGS5 (DOTA-DGlu-Ala-Tyr-Gly-Trp-(N-Me)Nle-Asp-1Nal-NH2), two new MG analogs were synthesized, by either introduction of a penta-DGlu moiety or depletion of the four N-terminal amino acids and introduction of a non-charged hydrophilic linker. Two CCK2R-expressing cell lines were used to demonstrate receptor interaction. Stability of the 177Lu-labeled peptide analogs was evaluated in human serum up to 24 h after incubation and in BALB/c mice up to 30 min after injection. The biodistribution profile and tumor targeting potential was evaluated in xenografted BALB/c nude mice. For both new MG analogs, the combination of strong receptor-specific cell interaction, high stability and enhanced tumor targeting could be demonstrated. Shortening of the peptide sequence lowered the absorption in the dose-limiting organs, whereas elongation increased uptake in renal tissue.

Strain-promoted azide–alkyne cycloaddition (SPAAC) is a straightforward and multipurpose conjugation strategy. The use of SPAAC to link different functional elements to prostate-specific membrane antigen (PSMA) ligands would facilitate the development of a modular platform for PSMA-targeted imaging and therapy of prostate cancer (PCa). As a first proof of concept for the SPAAC chemistry platform, we synthesized and characterized four dual-labeled PSMA ligands for intraoperative radiodetection and fluorescence imaging of PCa. Ligands were synthesized using solid-phase chemistry and contained a chelator for 111In or 99mTc labeling. The fluorophore IRDye800CW was conjugated using SPAAC chemistry or conventional N-hydroxysuccinimide (NHS)–ester coupling. Log D values were measured and PSMA specificity of these ligands was determined in LS174T-PSMA cells. Tumor targeting was evaluated in BALB/c nude mice with subcutaneous LS174T-PSMA and LS174T wild-type tumors using μSPECT/CT imaging, fluorescence imaging, and biodistribution studies. SPAAC chemistry increased the lipophilicity of the ligands (log D range: −2.4 to −4.4). In vivo, SPAAC chemistry ligands showed high and specific accumulation in s.c. LS174T-PSMA tumors up to 24 h after injection, enabling clear visualization using μSPECT/CT and fluorescence imaging. Overall, no significant differences between the SPAAC chemistry ligands and their NHS-based counterparts were found (2 h p.i., p > 0.05), while 111In-labeled ligands outperformed the 99mTc ligands. Here, we demonstrate that our newly developed SPAAC-based PSMA ligands show high PSMA-specific tumor targeting. The use of click chemistry in PSMA ligand development opens up the opportunity for fast, efficient, and versatile conjugations of multiple imaging moieties and/or drugs.

The first generation ligands for prostate-specific membrane antigen (PSMA)–targeted radio- and fluorescence-guided surgery followed by adjuvant photodynamic therapy (PDT) have already shown the potential of this approach. Here, we developed three new photosensitizer-based dual-labeled PSMA ligands by crucial modification of existing PSMA ligand backbone structures (PSMA-1007/PSMA-617) for multimodal imaging and targeted PDT of PCa. Various new PSMA ligands were synthesized using solid-phase chemistry and provided with a DOTA chelator for 111In labeling and the fluorophore/photosensitizer IRDye700DX. The performance of three new dual-labeled ligands was compared with a previously published first-generation ligand (PSMA-N064) and a control ligand with an incomplete PSMA-binding motif. PSMA specificity, affinity, and PDT efficacy of these ligands were determined in LS174T-PSMA cells and control LS174T wildtype cells. Tumor targeting properties were evaluated in BALB/c nude mice with subcutaneous LS174T-PSMA and LS174T wildtype tumors using µSPECT/CT imaging, fluorescence imaging, and biodistribution studies after dissection. In order to synthesize the new dual-labeled ligands, we modified the PSMA peptide linker by substitution of a glutamic acid into a lysine residue, providing a handle for conjugation of multiple functional moieties. Ligand optimization showed that the new backbone structure leads to high-affinity PSMA ligands (all IC50 < 50 nM). Moreover, ligand-mediated PDT led to a PSMA-specific decrease in cell viability in vitro (P < 0.001). Linker modification significantly improved tumor targeting compared to the previously developed PSMA-N064 ligand (≥ 20 ± 3%ID/g vs 14 ± 2%ID/g, P < 0.01) and enabled specific visualization of PMSA-positive tumors using both radionuclide and fluorescence imaging in mice. The new high-affinity dual-labeled PSMA-targeting ligands with optimized backbone compositions showed increased tumor targeting and enabled multimodal image-guided PCa surgery combined with targeted photodynamic therapy.

This document is intended as a supplement to the EANM “Guidelines on current Good Radiopharmacy Practice (cGRPP)” issued by the Radiopharmacy Committee of the EANM (Gillings et al. in EJNMMI Radiopharm Chem. 6:8, 2021 ). The aim of the EANM Radiopharmacy Committee is to provide a document that describes how to manage risks associated with small-scale “in-house” preparation of radiopharmaceuticals, not intended for commercial purposes or distribution.