Radiopharmaceutical research and development has been largely driven by the pursuit of high radiochemical yield (RCY) under optimized laboratory conditions. However, this emphasis on peak performance often overlooks a critical translational bottleneck: insufficient robustness under the rigid operational constraints encountered in automated synthesis modules (ASMs) and multi-center clinical production. Recognizing the multi-faceted nature of clinical translation, this perspective focuses specifically on the chemical design phase as the primary origin of translational reliability. Many radiolabeling systems achieve high RCY only within narrow operational windows and exhibit pronounced sensitivity to minor perturbations in process parameters, reflecting intrinsic chemical fragility driven by kinetic constraints, trace metal interference, and radiolysis at clinically relevant activity levels. We argue that radiochemical robustness, defined as consistent yield and purity across a multidimensional design space defined by critical process parameters, should be considered a primary design criterion. This approach aligns with Quality by Design (QbD) principles, evaluated through tolerance to environmental variability, reproducibility across scales and activity levels, and compatibility with standardized automated platforms. Integrating robustness-driven principles with traditional performance optimization serves as a critical de-risking strategy to reduce translational failure. Without compromising strict quality thresholds, this approach better aligns radiopharmaceutical development with stringent regulatory and current Good Radiopharmacy Practice (cGRPP) requirements, ensuring high-quality medicinal products for maximum patient benefit. Radiochemical robustness should be established as a co-equal criterion alongside biological performance during initial probe selection to ensure long-term translational viability.
The therapeutic radionuclide terbium-161 (161Tb) is a promising alternative to lutetium-177 (177Lu) for targeted radionuclide therapy, particularly for treating micrometastases. However, its clinical development is constrained by limited production capacity and the need for efficient separation from irradiated targets. This study developed a scalable, two-stage chromatographic process for producing no-carrier-added (n.c.a.) 161Tb, aiming to provide high-activity, high-purity material suitable for radiopharmaceutical development. High-purity n.c.a. [161Tb]TbCl₃ was obtained via sequential chromatography using a self-synthesized lanthanide (LN) resin and a commercial diglycolamide (DGA) resin. A single production batch yielded 42.38 ± 0.05
Cysteine cathepsin L (CatL) is emerging as a key biomarker of cancer progression, making it an attractive candidate for non-invasive molecular imaging. To monitor enzymatic activity directly, rather than merely protein expression, activity-based probes (ABPs) enable selective targeting of enzymes through covalent binding at their active sites. Despite vinyl sulfones are well-established irreversible inhibitors of cathepsins, their application as positron emission tomography (PET) ABPs remains unexplored. In this study we describe the design, synthesis, and evaluation of a novel class of 68Ga-labeled vinyl sulfone-based ABPs targeting CatL. A series of ABPs were synthesized based on the established vinyl sulfone scaffold K11777 (K777). Structure-activity relationship (SAR) analysis revealed that retaining a homophenylalanine (hPhe) residue at the P1 position resulted in greater CatL potency compared to its corresponding leucine (Leu) analogue. Evaluation of different linkers at the P3 position further revealed that polyethylene glycol (PEG) linkers preserved high CatL selectivity, whereas a piperazine linker increased off-target binding to cathepsin B (CatB). Based on these findings, two representative radiotracers were selected for further biological evaluation: the PEG2-linked [68Ga]CREANT-101 and the piperazine-linked [68Ga]CREANT-102. In vivo pharmacokinetic studies in healthy mice demonstrated that [68Ga]CREANT-102 exhibited prolonged blood circulation and high non-specific tissue uptake, whereas [68Ga]CREANT-101 displayed rapid systemic clearance and low background signal, supporting its selection for tumor imaging. In both U-87 MG and HT-29 xenograft models, [68Ga]CREANT-101 showed modest absolute tumor uptake in vivo. However, subsequent ex vivo analysis of tumor homogenates confirmed successful and specific engagement of active cathepsins within the target tissue. A novel class of 68Ga-labeled vinyl sulfone ABPs targeting CatL was successfully developed and preclinically evaluated. The lead tracer, [68Ga]CREANT-101, demonstrated high selectivity and a favorable background profile, but showed limited tumor accumulation in vivo. Ex vivo findings indicated that this limited tumor uptake was not attributable to insufficient target affinity, but rather to suboptimal cellular internalization and restricted tumor bioavailability, likely resulting from rapid systemic clearance and in vivo metabolic instability. Therefore, future optimization of this tracer class should focus on improving these pharmacokinetic properties to enhance in vivo tumor targeting.
Positron emission tomography (PET) is an advanced imaging modality that enables visualization of molecular and cellular processes, with PET probes serving as its critical core component. Molecular docking is a computational technique that predicts binding modes and interactions between ligands and their biological targets. Driven by breakthroughs in structural biology and computational technology, molecular docking has evolved into an indispensable auxiliary tool for modern PET probe development. This review introduces the fundamental principles of molecular docking, including core algorithms, scoring functions, and commonly used software packages. It then comprehensively examines its applications across the entire PET probe development pipeline: virtual screening for lead compound discovery; rational structural optimization along three complementary dimensions (reinforcing favorable interactions, eliminating unfavorable factors, and rational design of radiolabeling sites); and widespread retrospective analysis of binding modes. Furthermore, it addresses the application of molecular docking in systematic evaluation of existing probes, with a focus on tau-targeted tracers, where it has been used to compare binding sites, subtype selectivity, and off-target mechanisms across multiple probes and distinct tau fibril isoforms. This review illustrates the utility of molecular docking as a comprehensive tool in PET probe development. Although current applications remain predominantly retrospective analysis, with continuous breakthroughs and deep integration of technologies such as high‑precision structure prediction and GPU‑accelerated ultra‑large‑scale screening, molecular docking is poised to become a key enabler for the rational development of PET probes.
Isotopic exchange with fluorine-18 offers a conceptually simple approach to positron emission tomography (PET) tracer development, as the target compound itself serves as the labeling precursor. In this study, we investigated the influence of functional groups and substituent position on the efficiency of isotopic exchange with fluorine-18. A total of 19 fluorobenzene derivatives were screened under standardized conditions, in dimethyl sulfoxide (175 °C) and dimethylformamide (150 °C) as solvents. Only a subset of compounds underwent efficient exchange; electron-withdrawing substituents strongly promoted isotopic exchange, with ortho and para isomers generally being more reactive than meta-analogues. The kinetics of the exchange in dimethyl sulfoxide were quantified for fluorobenzonitriles, nitrofluorobenzenes, and fluorobenzaldehydes. Nonlinear regression analysis indicated more rapid apparent exchange for the ortho- and para-substituted nitrile and nitro derivatives, together with higher apparent plateaus (≥84–94
Prostate-specific membrane antigen (PSMA)–targeting radiopharmaceuticals are widely used for positron emission tomography (PET) imaging of prostate cancer and are often grouped as a single diagnostic class in clinical guidelines. However, beyond PSMA selectivity, each radiopharmaceutical exhibits distinct in vivo behaviour, and the molecular interactions and binding kinetics underlying these differences remain incompletely understood. This study aimed to define the mechanistic determinants of ligand-specific behaviour among clinically used 18F-fluorinated radiotracers by integrating molecular simulations with real-time cellular binding assays. Molecular docking and molecular dynamics simulations were used to characterise the binding of the fluorinated PSMA radiopharmaceuticals [18F]PSMA-1007, [18F]DCFPyL, and [18F]JK-PSMA-7 within the PSMA active site, with particular attention to zinc coordination and water-mediated interactions. Relative binding energies were estimated using the Molecular Mechanics Poisson–Boltzmann Surface Area method. Real-time cellular binding kinetics were assessed using the LigandTracer system to estimate apparent association and dissociation rate constants. These experiments included the three fluorinated tracers and [68Ga]Ga-PSMA-11 as a widely used clinical reference. Competition assays with 2-(phosphonomethyl)pentanedioic acid were also performed. All three fluorinated radiopharmaceuticals investigated in silico bound within the PSMA active site through the conserved Glu–urea–Lys motif and maintained stable interactions with the binuclear zinc centre. Molecular simulations revealed ligand-specific differences in the occupation of the active-site funnel and showed substantial contributions from water-mediated hydrogen-bond networks, particularly for [18F]PSMA-1007. In cellular experiments, [68Ga]Ga-PSMA-11 exhibited the highest mean apparent association rate and the most pronounced decrease in cell-associated signal during the dissociation phase. In contrast, [18F]PSMA-1007 showed the lowest mean apparent association rate and no measurable signal decrease during the experimental dissociation period, whereas [18F]DCFPyL and [18F]JK-PSMA-7 displayed intermediate kinetic profiles. Competition assays yielded progressively higher apparent IC50 estimates from [68Ga]Ga-PSMA-11 to [18F]PSMA-1007. No measurable decrease in [18F]PSMA-1007-associated signal was observed during the 30-minute real-time competition experiment, even at the highest inhibitor concentration. Despite sharing a conserved PSMA-binding motif, clinically used PSMA radiopharmaceuticals exhibited distinct molecular interaction patterns and apparent cellular binding kinetics under the experimental conditions examined, taking into account the simplifications adopted for the design of the study and for the analysis of the results. These findings provide a mechanistic basis for tracer-specific behaviour and support further investigation of whether such differences influence clinical imaging characteristics.
Metal complexes play indispensable roles in modern medicine, underpinning diagnostic techniques such as magnetic resonance imaging and nuclear imaging, as well as targeted radionuclide therapy. Their clinical success relies on chelators that form thermodynamically stable and kinetically inert metal complexes, thereby minimizing the release of toxic free metal ions in vivo. Although acyclic chelators were among the first ligands used clinically, their limited kinetic inertness prompted the development of macrocyclic polyaminopolycarboxylate chelators, whose preorganized cavities confer exceptional complex stability. To enable site-specific conjugation to peptides, antibodies, and other biological targeting vectors, these macrocycles must be functionalized into bifunctional chelating agents. Despite their widespread application, a comprehensive overview of the synthetic methodologies for preparing these bifunctional chelators has been lacking. This review comprehensively surveys the synthetic strategies for macrocyclic polyaminopolycarboxylate chelators used in molecular imaging and targeted radionuclide therapy. Particular emphasis is placed on cyclen-, 1,4,7-triazacyclononane-, cyclam-, and cage-based platforms, including the DOTA, NOTA, TETA, PCTA, DiamSar, and AmBaSar families and their structurally related derivatives. The review covers the synthesis of the parent macrocycles and their selective functionalization into bifunctional chelators for conjugation to biological targeting vectors. It further examines the synthesis of derivatives bearing amino, thiol, maleimide, N-hydroxysuccinimide ester, alkyne, and benzyl functionalities, with particular emphasis on protecting-group strategies, regioselective functionalization, purification methods, and reaction conditions that govern regioselectivity and yield. By consolidating these methodologies, the review provides a practical framework for the rational design and synthesis of bifunctional chelators for diagnostic and therapeutic radiopharmaceutical applications. The selective functionalization of macrocyclic polyaminopolycarboxylate chelators relies on carefully designed protection and deprotection strategies, with orthogonal protecting groups such as tert-butyloxycarbonyl, carboxybenzyl, and tert-butyl esters playing key roles in obtaining regioisomerically pure intermediates. The introduction of diverse functional groups, including amines, thiols, maleimides, N-hydroxysuccinimide esters, and alkynes, enables efficient conjugation to biological targeting vectors through amide coupling, Michael addition, and click chemistry. Collectively, the synthetic methodologies summarized in this review provide a robust foundation for the development of next-generation bifunctional chelators and theranostic radiopharmaceuticals with expanded applications in precision molecular imaging and targeted radionuclide therapy.
Abstract Background Glucagon-like peptide-1 receptor (GLP-1R) is a clinically validated therapeutic target for the treatment of obesity and type 2 diabetes. GLP-1Rs expressed in the central nervous system (CNS) regulate appetite and are therefore particularly important in the context of weight loss. There is thus an emerging need for efficient and brain-penetrating Positron Emission Tomography (PET) technologies to study the distribution of GLP-1R in the CNS and facilitate the development of novel GLP-1R-targeted therapeutics. However, currently established GLP-1R PET tracers are peptide-based and exhibit limited blood–brain barrier (BBB) penetration, restricting their use for imaging central GLP-1R expression. V-0219, a small molecule GLP-1R positive allosteric modulator, represents a potential scaffold for the development of BBB-penetrating PET tracers targeting incretin receptor systems in the brain. Results Here, we report the radiosynthesis and preclinical evaluation of [ 11 C]Methyl-V-0219, a carbon-11 labelled analogue of V-0219. [ 11 C]Methyl-V-0219 was synthesized using a Pd(0)-mediated Suzuki–Miyaura coupling reaction and obtained with a radiochemical yield of 36 ± 18% (n = 16) and radiochemical purity of 98.6 ± 1.6% (n = 16). In vitro autoradiography demonstrated retained binding to GLP-1R–positive tissues, although binding to glucose-dependent insulinotropic polypeptide (GIP) and glucagon (GCG) receptors was also observed. In vivo PET imaging was performed in rats and pigs and compared with the well-established GLP-1R tracer [ 68 Ga]Ga-DO3A-Exendin-4. Dynamic PET imaging demonstrated rapid brain uptake of [ 11 C]Methyl-V-0219 followed by progressive washout, indicating BBB penetration but limited retention in the brain. In contrast to [ 68 Ga]Ga-DO3A-Exendin-4, [ 11 C]Methyl-V-0219 did not demonstrate detectable retention in GLP-1R–rich tissues, including the pituitary gland and pancreas. Instead, prominent uptake was observed in the liver, intestines, and glandular tissues, consistent with hepatobiliary clearance and nonspecific accumulation of a lipophilic compound. Furthermore, blocking and competition studies did not alter tracer brain kinetics, suggesting no detectable displaceable binding in vivo. Conclusion Taken together, these findings demonstrate the feasibility of developing small–molecule–based PET tracers targeting incretin receptor systems, while highlighting the challenges associated with achieving sufficient in vivo specificity for brain GLP-1R imaging.
Many high-risk prostate cancer (PCa) patients need to undergo 99mTc-based bone scintigraphy as part of conventional diagnostics to assess metastasis to the bone. Besides, targeting the prostate-specific membrane antigen (PSMA) has proven to be very effective for theranostic approaches in PCa in recent years. Therefore, we recognized the high clinical potential of a dual-tracer approach, simultaneously targeting PSMA-expressing lesions and PSMA-negative bone anomalies. In a preclinical PCa model, we aimed at concurrent administration and dual-isotope single-photon emission computed tomography (SPECT) of two radiotracers labeled either with Indium-111 (111In) or Technetium-99m (99mTc). Separate depiction of the signals should illustrate the different biodistribution of [111In]In-PSMA-I T and 99mTc-3,3-diphosphono-1,2-propanodicarboxylic acid ([99mTc]Tc-DPD) in the animal. Temporally parallel radiosyntheses and analysis of [111In]In-PSMA-I T and [99mTc]Tc-DPD were successfully established. Co-incubation studies were conducted with the radioligand stock solutions as well as in vitro experiments using LNCaP cells. LNCaP xenograft-bearing CB17-SCID mice were injected almost simultaneously with [111In]In-PSMA-I T and [99mTc]Tc-DPD and scanned at different time points after injection (2–3 h and 23 h p.i.) on a Siemens Inveon µSPECT/CT. Thereafter, mice were subjected to biodistribution studies. Co-incubation experiments revealed no mutual detrimental effects on radioligand purities. In vitro studies confirmed that the presence of DPD has no influence on [111In]In-PSMA-I T binding and internalization into PSMA-expressing LNCaP cells. By application of three different acquisition windows (99mTc: 126–154 keV, 111In: 221–269 keV, open: 80 - 270 keV) radionuclide-specific uptake could be observed and distinguished in the PSMA-expressing xenografts ([111In]In-PSMA-I T) and in the bones ([99mTc]Tc-DPD). Biodistribution data corroborated these results. Dual-tracer SPECT imaging of PSMA-positive xenografts and PSMA-negative bone structures with [111In]In-PSMA-I T and [99mTc]Tc-DPD, respectively, was successfully realized in this preclinical approach. The positive results of this proof-of-concept study indicate a potentially prompt and successful transfer to clinical settings in which PSMA− bone lesions can be identified besides the PSMA+ primary tumor and metastases in a single SPECT/CT scan. This in turn could reduce scan time, CT dose and costs per patient, and provide important insights into tumor biology and improve stratification and treatment of PCa patients.
Radioactive isotopes, such as Indium-111 (111In), are pivotal for highly sensitive noninvasive imaging and targeted radiotherapy. However, achieving stable in vivo retention remains a critical barrier. Conventional nanocarriers typically rely on surface-bound chelators, exposing the isotopes to biological transchelation and detachment, while potentially altering the carrier’s surface properties and pharmacokinetics. To address these limitations, this study evaluates a polyionic complex micelle (PICm) platform designed to encapsulate 111In within a crosslinked core using DOTA chelation. This approach aims to enhance radiochemical stability while preserving the micelle’s native, engineered surface characteristics. DOTA-functionalized PICm were fabricated through electrostatic self-assembly of PEG-based polymers and subsequent core crosslinking. The resulting micelles exhibited a highly uniform size (32.6 ± 6.3 nm) and a modestly negative surface charge (− 4.9 ± 1.0 mV). Stability assays demonstrated that under the acidic conditions required for 111In chelation (pH 5.5), the micelles undergo reversible size swelling driven by polymer protonation without aggregating or collapsing, confirming that core crosslinking successfully maintains micellar integrity. In vivo biodistribution in CT26 tumor-bearing mice revealed that 111In-loaded PICm displayed prolonged systemic presence, with tumor accumulation peaking at 2.3 ± 0.4
Terbium-161 (161 Tb) is an emerging therapeutic radionuclide combining β⁻ emission with a high yield of Auger electrons and conversion electrons, enabling enhanced localized dose deposition. However, its widespread clinical adoption is limited by inefficient target utilization and multistep post-irradiation processing. This study aimed to develop a simplified high-yield production and direct chloride-based purification workflow for clinical-grade terbium-161 using thin-layer enriched gadolinium-160 targets under limited-resource reactor conditions. Terbium-161 was produced by neutron irradiation of enriched Gadolinium-160 targets fabricated as conventional oxide powder and novel thin-layer chloride films at a thermal neutron flux of 1.5 × 1014 n cm⁻2 s⁻1 for 24 h in Pakistan Atomic Research Reactor-1 (PARR-I). Post-irradiation separation of terbium was carried out using LN resin employing HCl and HNO₃ media, and compared with α-hydroxyisobutyric acid (α-HIBA) cation-exchange chromatography. Radionuclidic purity was assessed by HPGe γ spectrometry, while radiochemical purity and labeling efficiency with DOTA conjugated Peptides were evaluated using ITLC and HPLC. Thin-layer targets significantly enhanced neutron utilization, compared to oxide targets due to reduced self-shielding of target. The specific activities achieved across three production batches (34.25–79.01 MBq/mg) demonstrated an approximately 2.3-fold higher specific activity with 160GdCl3 layered targets than bulk Gd₂O₃ targets. LN–HCl separation provided superior recovery (91.4
Multiple new Positron Emission Tomography (PET) radiopharmaceuticals (RPhs) have recently been approved by the U.S. Food and Drug Administration (FDA). Despite the generally accepted safety profile of PET RPhs supported by millions of doses administered per year, the microdoses used in most injections, and the low incidence of adverse events (AEs), there appear to be elevated concerns from regulatory authorities regarding the safety of PET RPhs in recent years. There have been prior reports on the prevalence of AEs from RPhs, but most of them are outdated and do not reflect the current state-of-the-art and best practices. To address the gap, this retrospective study assesses risks associated with PET RPhs by reviewing AEs reported to the FDA Adverse Events Reporting System (FAERS) since the implementation of the PET good manufacturing practice (GMP) regulations in 21 CFR 212 in 2012. Data for AEs reported for approved PET RPhs were collected from the FAERS database. Data were collected during December 2025 for AEs reported between 2012 and 2024. FDA approved PET RPhs were searched using all known names for each drug. For each RPh, the following tables were downloaded from the database: (a) case count by received year; (b) cases by reaction; and (c) listing of cases. Tables downloaded for each radiopharmaceutical were combined, and duplicated entries removed. AE reports were then categorized based on the manufacturing regulatory oversight and, for the purposes of this report, we focused on AEs reported for 10 PET RPhs manufactured under 21 CFR 212. Almost 24 million adverse event reports for all drug products were transmitted to FDA between 2012 and 2024. Of these, 932 (or 0.0039
Overexpression of fibroblast activation protein (FAP) in cancer-associated fibroblasts enables a highly selective targeting strategy using FAP inhibitors. However, radiolabeled small-molecule FAP inhibitors (FAPIs) show limited tumor retention, restricting therapeutic applications. Multimerization represents a promising strategy to enhance tumor uptake and retention through polyvalent binding. Here, we report the rational design and evaluation of dimeric FAP-targeted radiopharmaceuticals for theranostic applications. Based on the (4-quinolinoyl)-glycyl-2-cyanopyrrolidine (QCP) scaffold, a series of monomeric FAPIs were assembled into dimeric radioligands using three distinct tripod-based platforms and equipped with a DOTA-GA chelator for efficient radiolabeling with indium-111 and lutetium-177; systematic in vitro and in vivo evaluation of binding affinity, stability, cellular uptake, and tumor targeting identified eFAP-51 and eFAP-52 as lead candidates, both showing high radiochemical yield and purity, favorable stability, and sub-nanomolar affinity for FAP with strong selectivity over DPP4. Further studies demonstrated superior tumor targeting and pharmacokinetics for eFAP-51 compared with the clinical reference FAPI-46, with [111In]In-eFAP-51 exhibiting more than 4-fold higher tumor uptake at 48 h post-injection (3.2 ± 0.5 vs. 0.71 ± 0.1
[11C]Phosgene is a known but underutilised labelling synthon in PET-tracer development. The production of [11C]phosgene faces many problems such as difficulties in achieving high molar activity, regular key component replacement and complexity of the synthesis, thereby hindering its wider use. In this paper, a robust and semi-automated synthesis route for production of [11C]phosgene from [11C]CO2 was developed to solve issues restricting the use of [11C]phosgene. The high molar activity [11C]phosgene was then used for the labelling of biologically important molecules as a proof of concept. [11C]Phosgene was synthesized from [11C]CO by UV-mediated photochemical chlorination with chlorine gas. [11C]CO was produced by the fluoride-activated disilane reduction of cyclotron-produced [11C]CO2. [11C]Phosgene was produced with satisfactory radiochemical yield (13 ± 1 GBq at EOS, 30 ± 4
18F-Labeled (E)-1-fluoro-3-((2-(4-(5-(methylamino)pyrazin-2-yl)but-1-en-3-yn-1-yl)benzo[d]thiazol-6-yl)oxy)propan-2-ol ([18F]SPAL-T-06) and (E)-1-fluoro-3-((2-(4-(6-(methylamino)pyridine-3-yl)but-1-en-3-yn-1-yl)benzo[d]thiazol-6-yl)oxy)propan-2-ol ([18F]C05-05) are positron emission tomography (PET) imaging agents for α-synuclein pathologies. Recently, we demonstrated the utility of PET imaging with [18F]SPAL-T-06 for α-synuclein pathologies in patients with multiple system atrophy and with [18F]C05-05 for α-synuclein pathologies in Parkinson’s disease and related disorders. In this study, to establish [18F]SPAL-T-06 and [18F]C05-05 as PET radiopharmaceuticals for clinical investigations, we developed routine radiosynthetic procedures using an 18F-labeling synthesizer and performed quality control (QC) testing of these two PET radiopharmaceuticals. [18F]SPAL-T-06 and [18F]C05-05 were synthesized by direct 18F-fluorination of their respective tetrahydropyranyl-protected tosylated precursors, followed by deprotection, using an 18F-labeling synthesizer. The non-decay-corrected radiochemical yields of [18F]SPAL-T-06 and [18F]C05-05 from 18F− at the end of synthesis (EOS) were 11 ± 4.6
Analytical data for 225Ac-labeled radiopharmaceuticals are currently obtained using thin-layer chromatography and high-performance liquid chromatography, the latter followed by offline fraction collection and delayed gamma-counting after secular equilibrium with gamma-emitting daughters has been reached. These procedures are slow and do not depict the distribution of daughter radionuclides. An inline HPLC detection setup combining liquid scintillation detection and LaBr3-based gamma-spectroscopy is presented to enable faster and more informative analysis of a 225Ac radiopharmaceutical. Four chromatographic signals were identified corresponding to uncomplexed 221Fr with at most a minor contribution of free 213Bi, [213Bi]Bi-PSMA I T, [225Ac]Ac-PSMA I T, and [209Pb]Pb-PSMA I T. The retention times corresponded to those of the non-radioactive analogs determined by UV detection. Assignments were confirmed by fraction collection followed by gamma-spectroscopy and beta liquid scintillation counting. The distribution of these species changed upon heating the reaction mixture or after addition of another ligand. The combined detection setup enables rapid observation of isotope distribution and daughter behavior during chromatographic analysis and avoids fraction handling by operating as a closed system, thereby reducing contamination risk and radiation exposure. This approach may contribute to improved quality control procedures for 225Ac radiopharmaceuticals and other alpha-emitting radionuclides. At the present stage, however, it should be regarded as a qualitative to semi-quantitative proof-of-principle.
Hitherto, clinically applied 99mTc-based cholecystokinin-2 receptor (CCK-2R) ligands suffer from limited in vivo stability. Therefore, we focused on the development of pharmacokinetically optimized 99mTc-labeled CCK-2R ligands for improved imaging of CCK-2R-overexpressing malignancies via single-photon emission computed tomography (SPECT). The novel CCK-2R ligands were designed based on the recently published compounds CCK-66, rhCCK-18 and rhCCK-84. CCK-2R ligands were prepared by solid-phase peptide synthesis (SPPS) and 99mTc-labeled via the tetraamine (N4) chelator (95 °C, 15 min) with final radiochemical purities ≥ 95
The clinical translation of molecularly targeted therapeutics and imaging agents represents a cornerstone of precision oncology, with the global theranostics market projected to exceed 25 billion by 2030. However, the development of theragnostic agents or diagnostic companions remains constrained by analytical bottlenecks in quality control, such as target-binding fraction, which are increasingly required by regulatory agencies as product release criteria during the translation process. Current methods, including enzyme-linked immunosorbent assay (ELISA), which require specialized resources or external CROs, or bead-based assays for radiolabeled compounds, which involve complex multi-step protocols; these limitations and others hamper their practical implementation in clinical manufacturing environments. Assay delays can postpone clinical trial initiation, increase development costs, and delay patient access to these agents. We have developed and validated a rapid, size-exclusion high-performance liquid chromatography (SE-HPLC) method for the determination of target-binding fractions of labeled biologics. The method separates the unbound biologic from the larger antigen-bound complex, allowing for rapid quantification. We validated the method using a panel of fluorescently labeled antibodies (panitumumab-IRDye800CW, nivolumab-IRDye800CW) and radiolabeled biologics ([18F]GEH200521, [18F]NOTA-ABY-030), assessing linearity, specificity, and concentration independence. The SE-HPLC method achieved excellent separation of bound and unbound species with a resolution (Rs) of 3.2. A strong linear relationship (R2 = 0.999) was observed between the antigen-to-antibody ratio and the measured binding fraction. The method demonstrated high specificity, with no binding detected with non-target antigens. The total assay and analysis time was less than 35 min, a significant improvement over traditional methods. SE-HPLC provides a rapid, specific, and cost-effective alternative to traditional binding fraction assessment methods, reducing quality control timelines from weeks/hours to minutes. The method’s compatibility with both fluorescent and radiolabeled biologics and integration with existing HPLC infrastructure represents a significant advancement in development workflows.
The biodistributions of radiometalated peptides and small molecules are greatly influenced by the charge conferred by the metal-chelator complex. Careful fine-tuning of this charge thus represents an attractive method to optimise pharmacokinetic properties. For this to be an effective strategy, numerous suitable chelators must be available for a given radiometal; each possessing a different net charge. Herein we report the synthesis of 2-(4,7,10-tris(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pentanedioic acid (DOTAMGA), a bifunctional chelator for the 203/212Pb theranostic pair containing a single negative charge, previously unattainable with reported radiolead chelators. DOTAMGA was incorporated into a radiopharmaceutical targeting the melanocortin 1 receptor (MC1R), a receptor highly expressed by melanomas, and evaluated in vivo against the commonly used radiolead chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic amide (TCMC). DOTAMGA was synthesised and conjugated to the MC1R targeting peptide MC1RL for subsequent investigation alongside TCMC-MC1RL. DOTAMGA-MC1RL and TCMC-MC1RL exhibited comparable affinity for MC1R in a series of competition binding assays with MC1R expressing cells. DOTAMGA-MC1RL was effectively labelled with 212Pb and 203Pb under standard radiolabelling conditions, and UV–Vis experiments demonstrated more rapid Pb2+ complexation than TCMC-MC1RL. Both 212Pb-labelled compounds remained > 90
