Positron emission tomography is a useful tool for quantitative imaging of neuroinflammation in human subjects; however, more suitable radiotracers are sought. One of the promising targets for imaging neuroinflammation is macrophage colony-stimulating factor 1 receptor (CSF1R) because of its expression on activated microglia. Several PET radiotracers for imaging CSF1R have been developed, however, a suitable CSF1R radiotracer radiolabeled with 18F is not yet available. We synthesized and performed preclinical evaluation of a new series of high-affinity, 18F-labeled PET radiotracers for imaging CSF1R. A new series of CSF1R ligands with nanomolar binding affinities and structural properties suitable for brain PET was synthesized. Five compounds of the series were radiolabeled with 18F to give the corresponding radiotracers [18F]1, [18F]3, [18F]4, [18F]5 and [18F]6. The radiotracers were tested in biodistribution experiments in control mice and demonstrated brain uptake above 1% injected dose per gram (%ID/g) of tissue, with [18F]3 exhibiting the highest uptake (12.5%ID/g) at 5min after injection, followed by rapid washout. In the liposaccharide (LPS)-induced murine model of neuroinflammation all new radiotracers demonstrated a significant increase in brain uptake corrected for blood radioactivity vs. baseline controls, with [18F]3 showing the greatest increase (97%). The highest binding potential value in LPS-treated mice was seen for [18F]3 (BP = 1.05). In control baboon PET studies [18F]3 manifested high brain uptake (standardized uptake value, SUV = 5.2), reversible kinetics in baseline scan and moderate specific binding (up to 20%) in the blocking scans with two CSF1R inhibitors, CPPC and GW2580. In mice and baboon, [18F]3 undergoes metabolism with formation of a radiometabolite with insignificant brain penetration. The [18F]3 formulation in saline with 7% ethanol was stable for 2.5h of storage. In vitro assay showed that 3 was selective for CSF1R (Ki = 5nM) against several kinases associated with inflammation. Radiation dosimetry in mice demonstrated that [18F]3 is safe for future translation to human subjects. A series of new PET radiotracers for imaging CSF1R was synthesized and tested in vitro and in animals. One member of the series, 2-[18F]fluoro-N-(4-(4-methylpiperazin-1-yl)-2-(piperidin-1-yl)phenyl)pyrimidine-4-carboxamide ([18F]3), manifests PET imaging properties that may be suitable for further testing on non-human primate models of neuroinflammation and translation to human subjects.

Several platinum radionuclides, including 191Pt, are promising candidates for DNA-targeted Auger electron radiotherapy; however, effective compound designs are needed for this application. In this study, we developed six novel 191Pt-labeled compounds and evaluated their DNA-targeting properties in PSMA-positive tumors. Six trithiol-Hoechst-PSMA (THP) conjugates that consist of a trithiol ligand for 191Pt labeling, Hoechst33258 for DNA binding, and a PSMA-targeted moiety were synthesized and labeled with 191Pt, achieving radiochemical yields of 60-80%. The six [191Pt]Pt-THP compounds were evaluated for DNA-binding ability and PSMA targeting specificity in vitro, and biodistribution experiments were performed with five of the compounds in mice bearing subcutaneous PSMA-positive and PSMA-negative xenografts. Among them, [191Pt]Pt-THP3-4 and [191Pt]Pt-THP3-8, in which Hoechst33258 is linked on one side of the trithiol ligand via a linear PEG linker and the PSMA-targeting moiety is linked on the other side via a C4 linker, had the best properties. These compounds maintained higher PSMA targeting specificity and DNA-binding ability both in vitro and in vivo than the other [191Pt]Pt-THP compounds, exhibiting similar DNA binding in PSMA-positive PC3 PIP tumors in vivo as in the cultured cells from which the xenograft was derived. This study highlighted the importance of the linkers between the three components (trithiol-Hoechst-PSMA) and demonstrated binding of intravenously administered [191Pt]Pt-THP3-4 and [191Pt]Pt-THP3-8 to DNA in PSMA-positive tumors. Our compound designs and findings could be a useful foundation for DNA-targeted Auger electron cancer therapy, especially with Pt radionuclides.

[11C]methyl-L-methionine ([11C]MET) and O-(2-[18F]fluoroethyl)-L-tyrosine ([18F]FET) are commonly used radiopharmaceuticals in positron emission tomography (PET) for diagnosis of brain tumors. The preparations can be released for human use after the determination of several quality parameters. The enantiomeric purity test is an integral component of the quality control (QC) procedure for these radiopharmaceuticals. In this context, the European Pharmacopoeia monographs recommend thin-layer chromatography (TLC) for the separation of optical isomers of radiolabeled amino acids. To enhance the accuracy and efficiency of the analysis, a liquid chromatographic method should be employed. The aim of this work was to evaluate ultrahigh-performance liquid chromatography (UPLC) for the determination of enantiomeric purity of [11C]MET and [18F]FET, with the goal of reducing analysis time. Additionally, a pre-column derivatization method was applied using o-phthaldialdehyde (OPA) and N‑isobutyryl‑L‑cysteine (IBLC), which are widely used for the separation of optical isomers of amino acids. In this work, two novel chromatographic methods were proposed for the determination of enantiomeric purity of [11C]MET and [18F]FET. The method development involved studying the effects of the type, composition, and acidity of the mobile phase, as well as the flow rate and column temperature on the separation of DD- and DL-diastereomers obtained from the reaction of amino acids and o-phthaldialdehyde and N-isobutyryl-L-cysteine derivatization reagents. Acquity BEH, CSH, and Kinetex XB stationary phases were tested with particle sizes ranging from 1.7 to 2.8μm. The finalized method used BEH C18 column (2.1 × 50mm, 1.7μm) with mobile phase consisting of 0.1% H3PO4 aqueous solution (A) and 0.1% H3PO4 in acetonitrile (B). In case of [11C]MET, the gradient elution was accomplished by increasing the acetonitrile content from 0 to 5% with 20min of gradient rate. For [18F]FET, the final acetonitrile ratio reached 40% over 25min. At a flow rate of 0.6ml/min the radiolabeled amino acids were separated within 10-20min with resolution > 1.5. The methods were tested in accordance with EANM guideline on the validation of analytical methods for radiopharmaceuticals. For linearity, r2 > 0.997 was obtained in the concentration range of 8-180 MBq/ml, the repeatability of %Area was < 5% (RSD%), recovery ranged from 101.8 to 105.4%, and the limit of quantitation (LOQ) was between 9 and 13 MBq/ml. The novel UPLC methods meet the Ph. Eur. monograph specifications and validation requirements. The pre-column derivatization reversed-phase (RP) chromatographic protocols are suitable for determining the enantiomeric purity of [11C]MET and [18F]FET radiopharmaceuticals and can be integrated into the quality control system.

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%, 70%, and 97% of the final product. A PC3-PIP patient-derived xenograft mouse model was used to investigate in vivo behavior of the formed impurity, followed by SPECT/CT imaging and biodistribution. When evaluating radio-HPLC methods for the impurity detection, the European Pharmacopoeia method (with a phosphate buffered eluent) shows no significant difference in radiochemical purity, an increased peak separation but decreased recovery (< 75%). The inter-laboratory comparison demonstrated the ability to measure the radiolysed radiopharmaceutical across multiple hospitals, with a maximum standard deviation of 6.6%. The in vivo SPECT/CT and biodistribution results show that as ingrowth of impurities caused by radiolysis increasing the tumor-to-kidney ratio is decreasing (97%/50% RCP, p < 0.05). The inter-laboratory study confirms that impurities caused by radiolysis can be measured accurately across different institutions, ensuring consistency and reliability in clinical diagnostics and therapy, contributing to improved patient safety. Phosphate buffered HPLC-methods showed no significant benefits in quality control. Increased impurities caused by radiolysis lead to lower tumor-to-kidney ratios, with a decrease in specific tumor binding.

Understanding the interaction between radiopharmaceuticals and human serum albumin (HSA) is essential for optimizing pharmacokinetics and therapeutic efficacy. This study evaluated the binding properties of natLu-FAPi-46 and natLu-FAP-2286 to HSA using theoretical and experimental techniques. Docking results revealed moderate affinities for natLu-FAPi-46 (- 9.7kcal/mol) and natLu-FAP-2286 (- 7.8kcal/mol), correlating with their lower blood retention (0.43% and 0.03% I.A./g at 4h p.i.). Comparative docking of albumin-binding derivatives of FAPi-46 showed stronger binding, consistent with increased blood retention. Experimental analyses (fluorescence quenching, circular dichroism, and cyclic voltammetry) confirmed complex formation and conformational changes in HSA, validating the computational findings. Together, the computational and experimental results underscore the importance of albumin-binding in shaping the pharmacokinetic properties of FAP-targeted radiopharmaceuticals. Strategic optimization of albumin-binding linkers may improve stability, circulation time, and overall therapeutic performance in next-generation FAP-based agents.

Fibroblast activation protein (FAP) is a pan-cancer target. Its selective expression on the majority of solid tumors with minimal to absent expression in healthy tissues positions FAP as a promising target for radiotheranostic applications. Nanobodies (Nbs) have unique characteristics, including small size, high affinity, stability, and ease of modification, making them ideal candidates for cancer diagnostics and targeted radiotherapeutics. Llama-derived Nbs were generated and screened against full-length FAP, with three unique candidates selected from the library for further characterization. The lead candidate Nb159 was engineered for site-specific radiolabeling with 89Zr for PET imaging and with 177Lu coupled with PEG for therapeutic evaluation in mice bearing FAP-positive U87 tumor xenografts. Nb159 exhibited exceptional picomolar binding affinity to FAP with stable interaction and slow dissociation. PET imaging with [89Zr]Zr-Nb159 demonstrated specific tumor uptake, peaking at 1 h post-injection, with rapid renal clearance and minimal uptake in non-target organs. A competitive binding study confirmed its specificity to FAP on U87 tumors, as pre-injection with a tenfold molar excess of unlabeled Nb159 reduced tumor uptake by ~ 55% (3.78 ± 0.50 to 1.67 ± 0.26%ID/g). PEGylation of Nb159 improved its pharmacokinetic profile, yielding prolonged tumor accumulation and significantly reduced renal retention when co-injected with lysine. PET imaging further demonstrated target-specific uptake in FAP-positive U87 xenografts, which exhibited higher signal than FAP-negative HCT116 tumors, with SUVmean at 48h of 0.45 ± 0.04 versus 0.09 ± 0.01 (P < 0.0001). In the therapeutic study, [177Lu]Lu-PEG-Nb159 demonstrated significant tumor growth inhibition with no observable toxicity. Mice treated with a single dose of [177Lu]Lu-PEG-Nb159 survived significantly longer compared to either [177Lu]Lu-DOTA (23 days, P < 0.001, HR: 0.06107) or vehicle (21 days, P < 0.0001, HR: 0.04017). The lead candidate Nb159 holds promise as a versatile platform for FAP-targeted radiotheranostics, with [89Zr]Zr-Nb159 serving as an effective companion diagnostic and [177Lu]Lu-PEG-Nb159 demonstrating promising therapeutic potential. These findings support further development of Nb159-based radiopharmaceuticals for treatment of FAP positive tumors.

[211At]PSMA-5 is a novel α-emitting therapeutic agent designed to target prostate-specific membrane antigen (PSMA), which is overexpressed in metastatic castration-resistant prostate cancer (mCRPC). Unlike β-emitting radioligands, [211At]PSMA-5 delivers highly localized cytotoxicity while minimizing damage to surrounding normal tissues. To enable clinical application, the objective of this study is to scale up the lab-scale synthesis to an automated manufacturing process that ensures high reproducibility and sufficient radioactivity for human administration. We developed and optimized a scalable automated synthesis method for [211At]PSMA-5 using the COSMiC-Mini VTRSC2 automated synthesizer. Optimization involved evaluating the recovery efficiency of 211At from the cold trap and reaction conditions, followed by automated synthesis under investigational new drug Good Manufacturing Practice conditions. Quality control of the synthesized [211At]PSMA-5 included assessment of radiochemical purity, radionuclide identity, impurity profile and sterility. Optimization with sodium hydrogen carbonate (7%) achieved over 90% recovery of 211At from the cold trap, and labeling rate of up to 93% were obtained using glass reaction vessels with stirring at 95°C. Three automated syntheses were conducted using irradiated Bi targets containing 211At produced at two different facilities. Consistent radiochemical yield (approximately 30%) and high radiochemical purity (96 ± 1%) were achieved. Additional quality control confirmed the absence of impurities such as 210At, Bi residues, and iodide, as well as sterility and chemical stability suitable for intravenous administration. This study successfully established an automated, scalable production process for [211At]PSMA-5 that meets clinical-grade quality requirements, enabling stable and reproducible manufacturing for investigator-initiated clinical trials in mCRPC. Equivalent radiochemical yields and consistent quality were obtained using irradiated Bi targets from two cyclotron facilities (RCNP and RIKEN), demonstrating site-independent robustness. This flexible system ensures a reliable supply and resilience to unexpected cyclotron downtime, representing a significant step toward clinical application of 211At-based PSMA-targeted alpha therapy.

[11C]Acetate and [11C]acetoacetate are PET radiotracers widely used to assess oxidative metabolism and ketone body utilization, respectively. This study aimed to establish robust, high-yield syntheses of both tracers using a GE TRACERlab FX2 C module, with an emphasis on improving radiochemical purity (RCP), radiochemical yield (RCY), optimizing operational parameters, and developing accurate quality control methods. [11C]Acetate was synthesized via Grignard carboxylation using [11C]CO2 and purified with a cartridge-based system. [11C]Acetoacetate was produced via in-loop [11C]CO2 carboxylation of a lithium enolate precursor, followed by semi-preparative reversed-phase HPLC purification. Quality control was performed by reported ion-exchange chromatography (IEX-HPLC) and novel reversed-phase HPLC (RP-HPLC). A systematic literature review was conducted to evaluate prior quality control methods for [11C]acetoacetate. Omission of helium flow during [11C]CO2 trap bake-out significantly improved activity recovery from the [11C]CO2 trap (from 63 to 89%) and reduced release time (from 4.8 to 3.1min). [11C]Acetate and [11C]acetoacetate were synthesized with mean isolated activities of 30.2GBq and 3.24GBq and mean RCPs of 96.9% and 97.1%, respectively. The final formulations met all European Pharmacopoeia criteria. While the widely-used IEX-HPLC method failed to differentiate [11C]acetate from [11C]acetoacetate, the newly-developed RP-HPLC method enabled unambiguous separation. Literature analysis revealed that most published studies on [11C]acetoacetate likely overlooked [11C]acetate as a radiochemical impurity due to insufficient analytical separation. Reliable synthesis protocols for [11C]acetate and [11C]acetoacetate were established on the GE TRACERlab FX2 C, with significant improvements in [11C]CO2 handling and product purification. Inclusion of the proposed RP-HPLC method enables a more accurate and specific assessment of RCP compared to IEX-HPLC alone and should be considered for a valid quality control of [11C]acetoacetate.

BackgroundActinium-225 (225Ac) based targeted alpha therapies (TAT) have emerged as a promising strategy for the treatment of several cancer types due to its favourable decay properties, including high linear energy transfer and short particle range, which enable precise tumour targeting. However, there are limited bifunctional chelators (BFCs) available for 225Ac. In this study, we aim to evaluate the potential of DEPA-based chelators for 225Ac-labelling.ResultsThe BFCs 3p-C-DEPA-NO2, 3p-C-DEPA-NCS, and 3p-C-DEPA-TFP-PEG4 were synthesized with high yield (≥ 86%) and purity (> 96%). Excellent radiochemical conversions (RCCs) were achieved for [225Ac]Ac-3p-C-DEPA-NO2 across a range of concentrations (0.7- 13.4 µg) with high RCC’s (93.7 to 96.8%) after 1 h at room temperature. Stability studies demonstrated that over 95% of this 225Ac-labelled complex remained intact after 6 days in human serum. The HPLC and bioanalyzer analysis of the immunoconjugates 3p-C-DEPA-TFP-PEG4-trastuzumab, DOTA-trastuzumab, 3p-C-DEPA-trastuzumab and macropa-trastuzumab showed 98% purity with less than 2% impurities. A RCC of 94.6% was obtained for [225Ac]Ac-3p-C-DEPA-trastuzumab, 93.5% for [225Ac]Ac-3p-C-DEPA-TFP-PEG4-trastuzumab, 80.9% for [225Ac]Ac-DOTA-trastuzumab, and 96.5% for [225Ac]Ac-macropa-trastuzumab after 2 h incubation at 37 °C. In PBS, high stability of [225Ac]Ac-3p-C-DEPA-TFP-PEG4-trastuzumab was observed (91.3 ± 4.3%), which is comparable to that of [225Ac]Ac-macropa-trastuzumab (81.9 ± 5.6%). In contrast, [225Ac]Ac-3p-C-DEPA-trastuzumab and [225Ac]Ac-DOTA-trastuzumab were less stable in PBS with only 48.3 ± 1.2% and 60.1 ± 0.6% intact tracer left after 10 d. There were no major significant differences between the biodistribution profile of [225Ac]Ac-3p-C-DEPA-trastuzumab, [225Ac]Ac-3p-C-DEPA-TFP-PEG4-trastuzumab, [225Ac]Ac-DOTA-trastuzumab and [225Ac]Ac-macropa-trastuzumab in all organs of interest (p > 0.05 for all organs).Conclusions3p-C-DEPA-TFP-PEG₄ demonstrated excellent potential as a bifunctional chelator for 225Ac, showing high radiolabelling efficiency under mild conditions and outstanding in vitro stability of the resulting 225Ac-labelled bioconjugate. Further preclinical studies are warranted to validate its therapeutic potential.Supplementary InformationThe online version contains supplementary material available at 10.1186/s41181-025-00408-w.

Radiochemical Purity (RCP) assessment is a fundamental quality control parameter in radiopharmaceutical production. Thin-Layer Chromatography (TLC) using a radio-TLC scanner is the most common method employed to determine RCP. However, commercial devices are often expensive and may not be accessible for research environments or budget-constrained laboratories. This study presents the development of a low-cost, open-source radio-TLC scanner utilizing a silicon photomultiplier-based scintillation detector and a linear actuator. The system was designed to scan TLC strips and provide quantitative analysis. Validation for analysis of technetium-99m labeled compounds followed regulatory guidelines and included assessment of background noise, linearity, repeatability, positional accuracy, and comparative analysis against a commercial scanner. The system demonstrated good analytical performance, and comparative testing revealed a strong agreement with the results obtained using a commercial radio-TLC scanner. The custom radio-TLC scanner represents a viable and affordable alternative for radiochemical purity assessment in radiopharmaceutical quality control while maintaining compliance with good manufacturing practice standards.