EJNMMI Radiopharmacy and Chemistry最新文献

Background

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.

Results

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 SUV_mean at 48 h 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).

Conclusions

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.

Background

[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.

Results

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.

Conclusions

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.

Background

[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.

Results

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 3.1 min). [¹¹C]Acetate and [¹¹C]acetoacetate were synthesized with mean isolated activities of 30.2 GBq and 3.24 GBq 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.

Conclusions

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.

Background

Actinium-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.

Results

The 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).

Conclusions

3p-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.

Background:

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.

Results:

The system demonstrated good analytical performance, and comparative testing revealed a strong agreement with the results obtained using a commercial radio-TLC scanner.

Conclusion:

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.

Background

Fibroblast activation protein inhibitor (FAPI)-based positron emission tomography (PET) radiopharmaceuticals have shown promise for imaging cancer-associated fibroblasts (CAFs), a key component of the tumor microenvironment. FAPI radiopharmaceuticals offer high tumor-to-background contrast and are not influenced by hyperglycemia. Among these, [¹⁸F]AlF-FAPI-74, labeled using the Al¹⁸F-method, offers logistical advantages over ⁶⁸Ga-labeled radiopharmaceuticals, including a longer physical half-life and suitability for large-scale, centralized production. This study reports a fully automated, efficient, and GMP-compliant synthesis of [¹⁸F]AlF-FAPI-74 using the Trasis AllInOne^® platform, alongside a refined isocratic radio-HPLC method that enhances fluorine-18 recovery and impurity resolution. Additionally, the impact of molar dose on [¹⁸F]AlF-FAPI-74 biodistribution is evaluated in a preclinical model and a clinical case-study highlighting the performance of [¹⁸F]AlF-FAPI-74 produced at high apparent molar activity is provided.

Results

The automated GMP-compliant production was validated in three independent runs, with an average decay-corrected yield and apparent molar activity of 50 ± 10% and 1124 ± 254 GBq/µmol at the end of synthesis, respectively. Total synthesis time was 30 min. Quality control used validated analytical methods, including an optimized radio-HPLC protocol, ensuring regulatory compliance and batch consistency. [¹⁸F]AlF-FAPI-74 was produced with a radiochemical purity ≥ 95% and demonstrated excellent radiochemical stability in its final formulation for at least 10 h post-synthesis, at a concentration of 2019 MBq/mL. The in vitro binding kinetics of [¹⁸F]AlF-FAPI-74 were evaluated in a HEK293 cell line stably expressing human FAP, demonstrating rapid, specific uptake and internalization, along with high target affinity. In vivo biodistribution studies revealed a dose-dependent “molar amount effect,” with doses > 30 nmol/kg yielding improved tumor-to-background ratios. This phenomenon was not observed in clinical imaging, where high molar activity supported excellent image contrast.

Conclusions

A robust, fully automated, and GMP-compliant production process for [¹⁸F]AlF-FAPI-74 is reported with high yield, purity, and stability, suitable for centralized production and distribution. The improved radio-HPLC method enhances quality control precision by accurately quantifying radiochemical and chemical purity. In vivo experiments confirmed fast tumor uptake of [¹⁸F]AlF-FAPI-74. While a clear mass effect on tumor-to-background ratios was observed in preclinical studies, high apparent molar activity resulted in excellent contrast in a clinical setting.

Background

Activated hepatic stellate cells (aHSCs) are the key cell population in the injured liver driving fibrogenesis. aHSCs express platelet-derived growth factor receptor beta (PDGFRß), which is absent from quiescent HSCs. PDGFRß is therefore an attractive target of PET tracers for imaging of fibrogenesis. Here, we present the pharmacological characterization of [⁶⁸Ga]Ga-DOTA-Cys-ATH001 in preparation for clinical translation and further confirm PDGFRß as a biomarker of activated HSCs in liver disease by single cell sequencing.

Methods

The expression of PDGFRß in subpopulations of HSCs was evaluated in scRNAseq datasets from both a mouse and human liver samples. DOTA-Cys-ATH001 was evaluated for affinity and mechanism of binding to PDGFRß. [⁶⁸Ga]Ga-DOTA-Cys-ATH001 was evaluated for binding in vitro in mouse and human liver biopsies. The in vivo stability, biodistribution, pharmacokinetics, dosimetry and microdosing toxicology were evaluated in rats and pigs.

Results

PDGFRß expression was specifically upregulated in activated HSCs. [⁶⁸Ga]Ga-DOTA-Cys-ATH001 could differentiate fibrotic liver from healthy liver. The binding co-localized with tissue areas positive for collagen deposition and PDGFRß immunostaining. Based on the microdosing toxicology study the no observed adverse effect level was at least 1000 µg/kg, suggesting that the intended clinical PET scan dose is safe for use. Dosimetry calculations of [⁶⁸Ga]Ga-DOTA-Cys-ATH001 predicted an effective dose in human amenable to repeated examinations.

Conclusions

The data presented here suggests that PDGFRβ PET imaging with [⁶⁸Ga]Ga-DOTA-Cys-ATH001 has potential for non-invasive detection of activated HSCs. Clinical translation of [⁶⁸Ga]Ga-DOTA-Cys-ATH001 is ongoing.

Background

Promising developments in Pb-212 radiopharmaceutical therapies have increased demand for Pb-203 diagnostic agents. Building on previous work from various isotope production facilities, this study optimized Pb-203 production from electrodeposited Tl targets at Brookhaven National Laboratory (BNL). The additional supply of Pb-203 may help meet growing preclinical and clinical demands.

Results

Two Tl targets were irradiated at the Brookhaven Linac Isotope Producer facility with 30 ± 1 MeV protons, measured using previously published cross section data. Distribution coefficients for Pb Resin in acetate media were investigated for both Na⁺ and K⁺ cations, where potassium acetate was ~ 4 times more effective at stripping Pb from the Pb Resin. The Tl electrodeposition was optimized to deposit 350 mg of Tl (~ 60 mg/cm²) on Au backing in under 6 h. The proposed separation process was completed in < 1.5 h and achieved > 98% and 92 ± 3% recovery of Tl and Pb, respectively, with an overall Tl-Pb separation factor of 6 × 10⁵. The experimentally measured half-life of Pb-203 was 52.4 ± 0.7 h, agreeing with 51.93 ± 0.02 h reported by the National Nuclear Data Center. The radioisotopic purity of the Pb fraction at 24 h post end of bombardment (EOB) from a 24 h irradiation was 66% Pb-203, 28% Pb-201, and 6% Pb-200. Following chemical separation, the Pb-203 produced in this work (21 MBq Pb-203 EOB) achieved apparent molar activities of 10 ± 5 and 0.9 ± 0.5 GBq/µmol for [²⁰³Pb]Pb-DOTAM and [²⁰³Pb]Pb-DO3A, respectively, decay corrected to EOB. Data derived from this work suggests BNL can produce > 10’s GBq Pb-203 with > 99% radiochemical and radioisotopic purity from Tl-205 for worldwide distribution.

Conclusions

The production and separation of Pb-203 from natural Tl target material was successfully demonstrated at BNL. Existing methods were adapted and optimized for the facilities at BNL. Results from this work will guide future large-scale Pb-203 production opportunities at BNL for clinical applications.

Background

The development of safer and more effective cell-based therapies requires robust methods for tracking cells in vivo. Positron emission tomography (PET) is a highly sensitive nuclear imaging technique capable of quantitatively tracking the in vivo fate of cells after administration. Here, we investigated a cell-labeling strategy based on metabolic glycoengineering (MGE) to introduce azides on the cell surface, followed by radiolabeling via the bioorthogonal perfluoroaryl azide (PFAA)-Staudinger reaction. We studied the metabolic incorporation of a tetraacetylated PFAA-derivatized mannosamine in Jurkat cells and evaluated whether three triarylphosphines bearing the REstrained Complexing Agent (RESCA), could be radiolabeled with aluminum-[F]fluoride (Al[¹⁸F]F) for use as bioorthogonal reagents in the PFAA-Staudinger ligation.

Results

Three novel triarylphosphines containing different linkers (hydrophilic ester, ethylenediamine, and cyclohexyl) between the phosphine moiety and the (+)-RESCA-chelator were synthesized and characterized. Kinetic assays showed that all compounds reacted with the PFAA-derivatized monosaccharide, exhibiting different reaction kinetics under the tested conditions. They were successfully radiolabeled with fluorine-18 under optimized mild conditions and provided key insights into the radiolabeling of small molecules bearing the RESCA-chelator. The ester derivative underwent rapid chemical decomposition while the ethylenediamine- and cyclohexyl-linked derivatives were more resistant, with the cyclohexyl analogue showing the highest stability against demetallation and/or defluorination. However, radiolabeling with Al[¹⁸F]F led to the oxidation of the phosphine moiety, with the major radiolabeled product corresponding to the oxidized form. At the same time, flow cytometry showed that the metabolic incorporation of the tetra-acetylated PFAA-derivatized mannosamine into Jurkat cells was substantially less efficient than that of the widely used tetra-acetylated N-azidoacetylmannosamine (Ac₄ManNAz) derivative at the equivalent concentration. Increased concentrations of the PFAA derivative compromised cell viability, which halted subsequent studies.

Conclusions

While the Al[¹⁸F]F radiolabeling of these (+)-RESCA-bearing small molecules offers high stability against demetallation and/or defluorination, the method cannot currently be applied to triarylphosphines due to oxidation during radiolabeling and requires further development. Among the synthesized compounds, the cyclohexyl-linked derivative exhibited the most favorable stability profile, making it a potential lead structure for future tracer development. Nevertheless, this study advanced our understanding of MGE with PFAA-derivatized monosaccharides and highlighted the need for further investigation before applying PFAA-Staudinger ligation to cell radiolabeling.

Background

Successful treatment of solid cancers relies on precise diagnosis, e.g. using noninvasive molecular imaging, followed by surgical removal and/or chemo/immunotherapy. Despite advances in pre-operative imaging, real-time intraoperative tools remain limited, which often results in high rates of tumor-positive margins and recurrence after tumor resection. To address this limitation, we aimed to develop multifunctional fibroblast activation protein alpha (FAP) targeting tracers for bimodal medical imaging, enabling both pre-operative noninvasive molecular imaging via positron emission tomography (PET) and optical visualization during intraoperative fluorescence-guided surgery.

Results

NODAGA-FAP647 and NODAGA-FAP800 targeting human FAP (hFAP) were synthesized bearing a (R)-NODAGA chelator and a fluorophore (AlexaFluor647 or IRDye800CW, respectively). Binding affinities and binding kinetics of both unlabeled and ^67/68Ga-labeled compounds were evaluated in vitro using HT1080 cells (hFAP-expressing and wild type, WT) along with respective frozen xenograft tissue sections. Using real-time binding, both compounds exhibited picomolar binding affinities to hFAP via radioactive/fluorescent detection. This was primarily driven by low dissociation rate constants in vitro. Pharmacokinetics and tumor uptake were evaluated via PET and fluorescence imaging in mice bearing xenografts from the same cells. In vivo, both compounds were rapidly distributed and accumulated in hFAP-expressing but not WT-HT1080 tumors within 10–20 min post-injection. Fluorescence imaging showed a similarly good and selective tumor uptake in the first two hours and a qualitatively visible difference compared to WT-HT1080 beyond 24 h. Both compounds were quickly cleared from normal tissue and excreted renally.

Conclusion

Two FAP-targeting bimodal ligands were synthesized and evaluated in vitro and in vivo, showing high specificity and selectivity, along with rapid and selective tumor accumulation. Their long tumor retention and high imaging contrast make them promising candidates for clinical translation.