Radiochemical Purity Research Articles

Improving Routine 89Zr-Immuno-PET Applications: Mild Iron Removal Can Favor the Use of Fe-DFO-N-suc-TFP Ester Over p-NCS-Bz-DFO.

A key aspect for the applicability of 89Zr-radioimmunoconjugates is inert modification and radiolabeling. The two commercially available bifunctional variants of the siderophore desferrioxamine (DFO), Fe-DFO-N-suc-TFP-ester and p-NCS-Bz-DFO, are most often used for clinical 89Zr-immuno-PET. The use of Fe-DFO-N-suc-TFP-ester is advantageous with regard to higher radiolysis stability and more facile assessment of radiochemical purity as well as chelator-to-mAb ratio. However, not all mAbs withstand the Fe-removal step at relatively low pH (4-4.5) using EDTA, which is needed after conjugation to allow 89Zr labeling. In this study, it was investigated whether hydroxybenzyl ethylenediamine (HBED) or the clinically approved deferiprone (DFP) can serve as an alternative for EDTA to establish a pH-independent mild method for Fe-removal and thereby broaden the applicability of Fe-DFO-N-suc-TFP-ester. Carrier-added [59Fe]Fe-DFO-N-suc-TFP-ester was used for mAb modification to enable direct tracking of the Fe-removal efficiency under various conditions. Whereas incomplete Fe-removal with HBED was observed at pH 5 or higher, Fe-removal with DFP was possible at a broad pH range (4-9). This provides a mild, pH-independent method for Fe-removal, improving the applicability and attractiveness of Fe-DFO-N-suc-TFP-ester for 89Zr-mAb preparation.

Radiolabelling and preclinical characterisation of [89Zr]Zr-Df-ATG-101 bispecific to PD-L1/4-1BB.

ATG-101, a bispecific antibody that simultaneously targets the immune checkpoint PD-L1 and the costimulatory receptor 4-1BB, activates exhausted T cells upon PD-L1 crosslinking. Previous studies demonstrated promising anti-tumour efficacy of ATG-101 in preclinical models. Here, we labelled ATG-101 with 89Zr to confirm its tumour targeting effect and tissue biodistribution in a preclinical model. We also evaluated the use of immuno-PET to study tumour uptake of ATG-101 in vivo. ATG-101, anti-PD-L1, and an isotype control were conjugated with p-SCN-Deferoxamine (Df). The Df-conjugated antibodies were radiolabelled with 89Zr, and their radiochemical purity, immunoreactivity, and serum stability were assessed. We conducted PET/MRI and biodistribution studies on [89Zr]Zr-Df-ATG-101 in BALB/c nude mice bearing PD-L1-expressing MDA-MB-231 breast cancer xenografts for up to 10 days after intravenous administration of [89Zr]Zr-labelled antibodies. The specificity of [89Zr]Zr-Df-ATG-101 was evaluated through a competition study with unlabelled ATG-101 and anti-PD-L1 antibodies. The Df-conjugation and [89Zr]Zr -radiolabelling did not affect the target binding of ATG-101. Biodistribution and imaging studies demonstrated biological similarity of [89Zr]Zr-Df-ATG-101 and [89Zr]Zr-Df-anti-PD-L1. Tumour uptake of [89Zr]Zr-Df-ATG-101 was clearly visualised using small-animal PET imaging up to 7 days post-injection. Competition studies confirmed the specificity of PD-L1 targeting in vivo. [89Zr]Zr-Df-ATG-101 in vivo distribution is dependent on PD-L1 expression in the MDA-MB-231 xenograft model. Immuno-PET with [89Zr]Zr-Df-ATG-101 provides real-time information about ATG-101 distribution and tumour uptake in vivo. Our data support the use of [89Zr]Zr-Df-ATG-101 to assess tumour and tissue uptake of ATG-101.

Manufacturing 6-[18F]Fluoro-L-DOPA via Flow Chemistry-Enhanced Photoredox Radiofluorination.

In this study, we introduce a practical methodology for the synthesis of PET probes by seamlessly combining flow chemistry with photoredox radiofluorination. The clinical PET tracer 6-[18F]FDOPA was smoothly prepared in a 24.3% non-decay-corrected yield with over 99.0% radiochemical purity (RCP) and enantiomeric excess (ee), notably by a simple cartridge-based purification. The flow chemistry-enhanced photolabeling method supplies an efficient and versatile solution for the synthesis of 6-[18F]FDOPA and for more PET tracer development.

Synthesis and Evaluation of a Novel PET Radioligand for Imaging Glutaminyl Cyclase Activity as a Biomarker for Detecting Alzheimer's Disease.

Several new lines of research have demonstrated that a significant number of amyloid-β peptides found in Alzheimer's disease (AD) are truncated and undergo post-translational modification by glutaminyl cyclase (QC) at the N-terminal. Notably, QC's products of Abeta-pE3 and Abeta-pE11 have been active targets for investigational drug development. This work describes the design, synthesis, characterization, and in vivo validation of a novel PET radioligand, [18F]PB0822, for targeted imaging of QC. We report herein a simplified and robust chemistry for the synthesis of the standard compound, [19F]PB0822, and the corresponding [18F]PB0822 radioligand. The PET probe was developed with 99.9% radiochemical purity, a molar activity of 965 Ci.mmol-1, and an IC50 of 56.3 nM, comparable to those of the parent PQ912 inhibitor (62.5 nM). Noninvasive PET imaging showed that the probe is distributed in the brain 5 min after intravenous injection. Further, in vivo PET imaging with [18F]PB0822 revealed that AD 5XFAD mice harbor significantly higher QC activity than WT counterparts. The data also suggested that QC activity is found across different brain regions of the tested animals.

[18F]AlF-NOTA-PCP2: a novel PET/CT tracer for enhanced PD-L1 heterogeneity imaging and comparative analysis with [18F]AlF-NOTA-WL12 in glioblastoma xenografts.

The unsatisfactory efficacy of PD-L1 antibodies in glioblastoma (GBM) is largely due to the temporal and spatial heterogeneity of PD-L1 expression. Molecular imaging can enhance understanding of the tumor immune microenvironment and guide immunotherapy. However, highly sensitive imaging agents capable of effectively visualizing PD-L1 heterogeneity are limited. This study introduces a novel PET tracer, offering improved imaging of PD-L1 heterogeneity in GBM xenografts, with a comparative analysis to [18F]AlF-NOTA-WL12. [18F]AlF-NOTA-PCP2 was synthesized with high purity and its affinity for PD-L1 was characterized using surface plasmon resonance (SPR) and cell binding assays. Its specificity for PD-L1 was evaluated both in vitro using various cell lines and in vivo with GBM xenograft models in NOD/SCID mice. PET/CT imaging was conducted to evaluate the tracer's biodistribution, pharmacokinetics, and ability to quantify tumoral spatial heterogeneity of PD-L1 expression. A focused comparative analysis between [18F]AlF-NOTA-PCP2 and [18F]AlF-NOTA-WL12 was conducted, examining binding affinity, biodistribution, pharmacokinetics, and imaging effectiveness in GBM xenografts. Additionally, human radiation dosimetry estimates compared the safety profiles of both tracers. [18F]AlF-NOTA-PCP2 demonstrated high radiochemical purity (> 95%) and a strong affinity for PD-L1, comparable to [18F]AlF-NOTA-WL12. In vitro and in vivo studies confirmed its specificity for PD-L1, with increased uptake in PD-L1 expressing cells and tumors. Toxicological profiles indicated no significant abnormalities in serum biochemical indicators or major organ tissues. MicroPET/CT imaging showed [18F]AlF-NOTA-PCP2's effectiveness in visualizing PD-L1 expression levels and spatial heterogeneity in GBM xenografts. Comparative studies revealed [18F]AlF-NOTA-PCP2's improved pharmacokinetic properties, including higher tumor-to-blood ratios and lower nonspecific liver uptake, as well as reduced radiation exposure compared to [18F]AlF-NOTA-WL12. [18F]AlF-NOTA-PCP2 distinguishes itself as an exceptionally sensitive PET/CT tracer, adept at non-invasively and accurately quantifying PD-L1 expression and its spatial heterogeneity in tumors, especially in GBM. Its favorable pharmacokinetic properties, safety profile, and high affinity for PD-L1 highlight its potential for enhancing the precision of cancer immunotherapy and guiding individualized treatment strategies. While promising, its clinical translation, especially in brain imaging, necessitates further validation in clinical trials.

Engineering PD-1-targeted small protein variants for in vitro diagnostics and in vivo PET imaging

BackgroundProgrammed cell death 1 (PD-1) belongs to immune checkpoint proteins ensuring negative regulation of the immune response. In non-small cell lung cancer (NSCLC), the sensitivity to treatment with anti-PD-1 therapeutics, and its efficacy, mostly correlated with the increase of tumor infiltrating PD-1+ lymphocytes. Due to solid tumor heterogeneity of PD-1+ populations, novel low molecular weight anti-PD-1 high-affinity diagnostic probes can increase the reliability of expression profiling of PD-1+ tumor infiltrating lymphocytes (TILs) in tumor tissue biopsies and in vivo mapping efficiency using immune-PET imaging.MethodsWe designed a 13 kDa β-sheet Myomedin scaffold combinatorial library by randomization of 12 mutable residues, and in combination with ribosome display, we identified anti-PD-1 Myomedin variants (MBA ligands) that specifically bound to human and murine PD-1-transfected HEK293T cells and human SUP-T1 cells spontaneously overexpressing cell surface PD-1.ResultsBinding affinity to cell-surface expressed human and murine PD-1 on transfected HEK293T cells was measured by fluorescence with LigandTracer and resulted in the selection of most promising variants MBA066 (hPD-1 KD = 6.9 nM; mPD-1 KD = 40.5 nM), MBA197 (hPD-1 KD = 29.7 nM; mPD-1 KD = 21.4 nM) and MBA414 (hPD-1 KD = 8.6 nM; mPD-1 KD = 2.4 nM). The potential of MBA proteins for imaging of PD-1+ populations in vivo was demonstrated using deferoxamine-conjugated MBA labeled with 68Galium isotope. Radiochemical purity of 68Ga-MBA proteins reached values 94.7–99.3% and in vitro stability in human serum after 120 min was in the range 94.6–98.2%. The distribution of 68Ga-MBA proteins in mice was monitored using whole-body positron emission tomography combined with computerized tomography (PET/CT) imaging up to 90 min post-injection and post mortem examined in 12 mouse organs. The specificity of MBA proteins was proven by co-staining frozen sections of human tonsils and NSCLC tissue biopsies with anti-PD-1 antibody, and demonstrated their potential for mapping PD-1+ populations in solid tumors.ConclusionsUsing directed evolution, we developed a unique set of small binding proteins that can improve PD-1 diagnostics in vitro as well as in vivo using PET/CT imaging.Graphical

Validation of HPLC and TLC analytical methods to determine radiochemical purity of 99mTc-cAbVCAM1-5, a new experimental radiotracer

Validation of HPLC and TLC analytical methods to determine radiochemical purity of 99mTc-cAbVCAM1-5, a new experimental radiotracer

Challenges in the solution phase synthesis of PSMA-11 and PSMA-617: organic ligands for radiopharmaceutical preparations in prostate cancer medication

Abstract Patient specific treatments for different cancers are currently being actively addressed through nuclear medicine. More recently, the identification of biomarker namely; prostate-specific membrane antigen (PSMA) expressed on the prostate cancer cell surface has been considered as a turning point in prostate cancer management using radiopharmaceuticals. In this treatment method, apart from radionuclide, organic ligands that target PSMA constitute an essential component. PSMA-11 and PSMA-617 are two important ligands that form the radiopharmaceuticals, [68Ga]Ga-PSMA-11, [177Lu]Lu-PSMA-617, which are currently powering the prostate cancer management, especially metastatic castration resistant prostate cancer (mCRPC) in most part of the world. Identification of efficient synthetic routes towards these highly expensive ligands is an important prerequisite to make this treatment modality more popular. In this account, the synthetic challenges that we circumvent during the solution phase synthesis of PSMA-11 and PSMA-617, through different chemical synthetic routes are demonstrated. Post-synthesis, both the ligands, PSMA-11 and PSMA-617 were successfully radiolabelled using 68Ga, and 177Lu, respectively, to generate corresponding labelled products [68Ga]Ga-PSMA-11, and [177Lu]Lu-PSMA-617, in good radiochemical purity.

Advancing 6-bromo-7-[11C]methylpurine to clinical use: improved regioselective radiosynthesis, non-clinical toxicity data and human dosimetry estimates

Background6-Bromo-7-[11C]methylpurine ([11C]BMP) is a radiotracer for positron emission tomography (PET) to measure multidrug resistance-associated protein 1 (MRP1) transport activity in different tissues. Previously reported radiosyntheses of [11C]BMP afforded a mixture of 7- and 9-[11C]methyl regioisomers. To prepare for clinical use, we here report an improved regioselective radiosynthesis of [11C]BMP, the results of a non-clinical toxicity study as well as human dosimetry estimates based on mouse PET data.Results[11C]BMP was synthesised by regioselective N7-methylation of 6-bromo-7H-purine (prepared under good manufacturing practice) with [11C]methyl triflate in presence of 2,2,6,6-tetramethylpiperidine magnesium chloride in a TRACERlab™ FX2 C synthesis module. [11C]BMP was obtained within a total synthesis time of approximately 43 min in a decay-corrected radiochemical yield of 20.5 ± 5.2%, based on starting [11C]methyl iodide, with a radiochemical purity > 99% and a molar activity at end of synthesis of 197 ± 130 GBq/μmol (n = 28). An extended single-dose toxicity study conducted in male and female Wistar rats under good laboratory practice after single intravenous (i.v.) administration of unlabelled BMP (2 mg/kg body weight) revealed no test item related adverse effects. Human dosimetry estimates, based on dynamic whole-body PET data in female C57BL/6J mice, suggested that an i.v. injected activity amount of 400 MBq of [11C]BMP will deliver an effective dose in the typical range of 11C-labelled radiotracers.Conclusions[11C]BMP can be produced in sufficient amounts and acceptable quality for clinical use. Data from the non-clinical safety evaluation showed no adverse effects and suggested that the administration of [11C]BMP will be safe and well tolerated in humans.

Development and first-in-human study of PSMA-targeted PET tracers with improved pharmacokinetic properties.

A series of new 68Ga-labeled tracers based on [68Ga]Ga-PSMA-617 were developed to augment the tumor-to-kidney ratio and reduce the activity accumulation in bladder, ultimately minimize radiation toxicity to the urinary system. We introduced quinoline group, phenylalanine and decanoic acid into different tracers to enhance their lipophilicity, strategically limiting their metabolic pathway through the urinary system. Their binding affinity onto LNCaP cells was determined through in vitro saturation assays and competition binding assays. In vivo metabolic study, PET imaging and biodistribution experiment were performed in LNCaP tumor-bearing B-NSG male mice. The most promising tracer was selected for first-in-human study. Four radiotracers were synthesized with radiochemical purity (RCP) > 95% and molar activity in a range of 20.0-25.5GBq/μmol. The binding affinities (Ki) of TWS01, TWS02 to PSMA were in the low nanomolar range (< 10nM), while TWS03 and TWS04 exhibited binding affinities with Ki > 20nM (59.42nM for TWS03 and 37.14nM for TWS04). All radiotracers exhibited high stability in vivo except [68Ga]Ga-TWS03. Micro PET/CT imaging and biodistribution analysis revealed that [68Ga]Ga-TWS02 enabled clear tumor visualization in PET images at 1.5h post-injection, with higher tumor-to-kidney ratio (T/K, 0.93) and tumor-to-muscle ratio (T/M, 107.62) compared with [68Ga]Ga-PSMA-617 (T/K: 0.39, T/M: 15.01) and [68Ga]Ga-PSMA-11 (T/K: 0.15, T/M: 24.00). In first-in-human study, [68Ga]Ga-TWS02 effectively detected PCa-associated lesions including primary and metastatic lesions, with lower accumulation in urinary system, suggesting that [68Ga]Ga-TWS02 might be applied in the detection of bladder invasion, with minimized radiation toxicity to the urinary system. Introduction of quinoline group, phenylalanine and decanoic acid into different tracers can modulate the binding affinity and pharmacokinetics of PSMA in vivo. [68Ga]Ga-TWS02 showed high binding affinity to PSMA, excellent pharmacokinetic properties and clear imaging of PCa-associated lesions, making it a promising radiotracer for the clinical diagnosis of PCa. Moreover, TWS02 with a chelator DOTA could also label 177Lu and 225Ac, which could be used for PCa treatment without significant side effects. The clinical evaluation of this study was registered On October 30, 2021 at https://www.chictr.org.cn/ (No: ChiCTR2100052545).

Small animal PET imaging with the 68Ga-labeled pH (low) insertion peptide-like peptide YJL-4 in a triple-negative breast cancer mouse model

BackgroundThe aim of this study was to prepare a novel 68Ga-labeled pH (low) insertion peptide (pHLIP)-like peptide, YJL-4, and determine its value for the early diagnosis of triple-negative breast cancer (TNBC) via in vivo imaging of tumor-bearing nude mice. The novel peptide YJL-4 was designed using a template-assisted method and synthesized by solid-phase peptide synthesis. After modification with the chelator 1,4,7‑triazacyclononane-N,N′,N″-triacetic acid (NOTA), the peptide was labeled with 68Ga. Then, the biodistribution of 68Ga-YJL-4 in tumor-bearing nude mice was investigated, and the mice were imaged by small animal positron emission tomography (PET).ResultsThe radiochemical yield and radiochemical purity of 68Ga-YJL-4 were 89.5 ± 0.16% and 97.95 ± 0.06%, respectively. The biodistribution of 68Ga-YJL-4 in tumors (5.94 ± 1.27% ID/g, 6.72 ± 1.69% ID/g and 4.54 ± 0.58% ID/g at 1, 2 and 4 h after injection, respectively) was significantly greater than that of the control peptide in tumors at the corresponding time points (P < 0.01). Of the measured off-target organs, 68Ga-YJL-4 was highly distributed in the liver and blood. The small animal PET imaging results were consistent with the biodistribution results. The tumors were visualized by PET at 2 and 4 h after the injection of 68Ga-YJL-4. No tumors were observed in the control group.ConclusionsThe novel pHLIP family peptide YJL-4 can adopt an α-helical structure for easy insertion into the cell membrane in an acidic environment. 68Ga-YJL-4 was produced in high radiochemical yield with good stability and can target TNBC tissue. Moreover, the strong concentration of radioactive 68Ga-YJL-4 in the abdomen does not hinder the imaging of early TNBC.

Fundamental evaluation regarding the relationship between albumin-binding and tumor accumulation of PSMA-targeting radioligands.

The marked success of prostate-specific membrane antigen (PSMA)-targeting radioligands with albumin binder (ALB) is attributed to the improvement of blood retention and tumor accumulation. [111In]In-PNT-DA1, our PSMA-targeting radioligand with ALB, also achieved improved tumor accumulation due to its prolonged blood retention. Although the advantage of ALBs is related to their reversible binding to albumin, the relationship between albumin-binding and tumor accumulation of PSMA-targeting radioligands remains unclear because of the lack of information about radioligands with stronger albumin-binding than ALBs. In this study, we designed and synthesized [111In]In-PNT-DM-HSA, a new radioligand that consists of a PSMA-targeting radioligand covalently bound to albumin. The pharmacokinetics of [111In]In-PNT-DM-HSA was compared with those of [111In]In-PNT-DA1 and [111In]In-PSMA-617, a non-ALB-conjugated radioligand, to evaluate the relationship between albumin-binding and tumor accumulation. The [111In]In-PNT-DM-HSA was prepared by incubation of [111In]In-PNT-DM, a PSMA-targeting radioligand including a maleimide group, and human serum albumin (HSA). The ability of [111In]In-PNT-DM-HSA was evaluated by in vitro assays. A biodistribution study using LNCaP tumor-bearing mice was conducted to compare the pharmacokinetics of [111In]In-PNT-DM-HSA, [111In]In-PNT-DA1, and [111In]In-PSMA-617. The [111In]In-PNT-DM-HSA was obtained at a favorable radiochemical yield and high radiochemical purity. In vitro assays revealed that [111In]In-PNT-DM-HSA had fundamental characteristics as a PSMA-targeting radioligand interacting with albumin covalently. In a biodistribution study, [111In]In-PNT-DM-HSA and [111In]In-PNT-DA1 showed higher blood retention than [111In]In-PSMA-617. On the other hand, the tumor accumulation of [111In]In-PNT-DA1 was much higher than [111In]In-PNT-DM-HSA and [111In]In-PSMA-617. These results indicate that the moderate reversible binding of ALB with albumin, not covalent binding, may play a critical role in enhancing the tumor accumulation of PSMA-targeting radioligands.

Diagnostic Positron Emission Tomography Imaging with Zirconium-89 Desferrioxamine B Squaramide: From Bench to Bedside.

ConspectusMolecular imaging with antibodies radiolabeled with positron-emitting radionuclides combines the affinity and selectivity of antibodies with the sensitivity of Positron Emission Tomography (PET). PET imaging allows the visualization and quantification of the biodistribution of the injected radiolabeled antibody, which can be used to characterize specific biological interactions in individual patients. This characterization can provide information about the engagement of the antibody with a molecular target such as receptors present in elevated levels in tumors as well as providing insight into the distribution and clearance of the antibody. Potential applications of clinical PET with radiolabeled antibodies include identifying patients for targeted therapies, characterization of heterogeneous disease, and monitoring treatment response.Antibodies often take several days to clear from the blood pool and localize in tumors, so PET imaging with radiolabeled antibodies requires the use of a radionuclide with a similar radioactive half-life. Zirconium-89 is a positron-emitting radionuclide that has a radioactive half-life of 78 h and relatively low positron emission energy that is well suited to radiolabeling antibodies. It is essential that the zirconium-89 radionuclide be attached to the antibody through chemistry that provides an agent that is stable in vivo with respect to the dissociation of the radionuclide without compromising the biological activity of the antibody.This Account focuses on our research using a simple derivative of the bacterial siderophore desferrioxamine (DFO) with a squaramide ester functional group, DFO-squaramide (DFOSq), to link the chelator to antibodies. In our work, we produce conjugates with an average ∼4 chelators per antibody, and this does not compromise the binding of the antibody to the target. The resulting antibody conjugates of DFOSq are stable and can be easily radiolabeled with zirconium-89 in high radiochemical yields and purity. Automated methods for the radiolabeling of DFOSq-antibody conjugates have been developed to support multicenter clinical trials. Evaluation of several DFOSq conjugates with antibodies and low molecular weight targeting agents in tumor mouse models gave PET images with high tumor uptake and low background. The promising preclinical results supported the translation of this chemistry to human clinical trials using two different radiolabeled antibodies. The potential clinical impact of these ongoing clinical trials is discussed.The use of DFOSq to radiolabel relatively low molecular weight targeting molecules, peptides, and peptide mimetics is also presented. Low molecular weight molecules typically clear the blood pool and accumulate in target tissue more rapidly than antibodies, so they are usually radiolabeled with positron-emitting radionuclides with shorter radioactive half-lives such as fluorine-18 (t1/2 ∼ 110 min) or gallium-68 (t1/2 ∼ 68 min). Radiolabeling peptides and peptide mimetics with zirconium-89, with its longer radioactive half-life (t1/2 = 78 h), could facilitate the centralized manufacture and distribution of radiolabeled tracers. In addition, the ability to image patients at later time points with zirconium-89 based agents (e.g. 4-24 h after injection) may also allow the delineation of small or low-uptake disease sites as the delayed imaging results in increased clearance of the tracer from nontarget tissue and lower background signal.

64Cu tumor labeling with hexadentate picolinic acid-based bispidine immunoconjugates.

Discussed are two picolinate appended bispidine ligands (3,7-diazabicyclo[3.3.1]nonane derivatives) in comparison with an earlier described bis-pyridine derivative, which are all known to strongly bind CuII. The radiopharmacological characterization of the two isomeric bispidine complexes includes quantitative labeling with 64CuII at ambient conditions with high radiochemical purities and yields (molar activities >200 MBq/nmol). Challenge experiments in presence of EDTA, cyclam, human serum and SOD demonstrate high stability and inertness of the 64Cu-bispidine complexes. Biodistribution studies performed in Wistar rats indicate a rapid renal elimination for both 64Cu-labeled chelates. The bispidine ligand with the picolinate group in N7 position was selected for further biological experiments, and its backbone was therefore substituted with a benzyl-NCS group at C9. Two tumor target modules (TMs), targeting prostate stem cell antigen (PSCA), overexpressed in prostate cancer, and the fibroblast activation protein (FAP) in fibrosarcoma, were selected for thiourea coupling with the NCS-functionalized ligand and lysine residues of TMs. Small animal PET experiments on tumor-bearing mice showed specific accumulation of the 64Cu-labeled TMs in PSCA- and FAP-overexpressing tumors (standardized uptake value (SUV) for PC3: 2.7±0.6 and HT1080: 7.2±1.25) with almost no uptake in wild type tumors.

Increasing Analytical Quality by Designing a Thin-Layer Chromatography Scanner Method for the Determination of the Radiochemical Purity of Radiopharmaceutical Sodium Iodide 131I Oral Solution.

The goal of this study was to apply the principles of analytical quality by design (AQbD) to the analytical method for determining the radiochemical purity (PQR) of the radiopharmaceutical sodium iodide 131I oral solution, utilizing thin-layer chromatography (TLC) with a radio-TLC scanner, which also enables the evaluation of product quality. For AQbD, the analytical target profile (ATP), critical quality attributes (CQA), risk management, and the method operable design region (MODR) were defined through response surface methodology to optimize the method using MINITAB® 19 software. This study encompassed the establishment of a control strategy and the validation of the method, including the assessment of selectivity, linearity, precision, robustness, detection limit, quantification limit, range, and the stability of the sample solution. Under the experimental conditions, the method parameters of the TLC scanner were experimentally demonstrated and optimized with an injection volume of 3 µL, a radioactive concentration of 10 mCi/mL, and a carrier volume of 40 µL. Statistical analysis confirmed the method's selectivity for the 131I iodide band Rf of 0.8, a radiochemical impurity IO3- Rf of 0.6, a linearity from 6.0 to 22.0 mCi/mL, and an intermediate precision with a global relative standard deviation (RSD) of 0.624%. The method also exhibited robustness, with a global RSD of 0.101%, a detection limit of 0.09 mCi/mL, and a quantification limit of 0.53 Ci/mL, meeting the prescribed range and displaying stability over time (at 0, 2, and 20 h) with a global RSD of 0.362%, resulting in consistent outcomes. The development of a method based on AQbD facilitated the creation of a design space and an operational space, with comprehensive knowledge of the method's characteristics and limitations. Additionally, throughout all operations, compliance with the acceptance criteria was verified. The method's validity was confirmed under the established conditions, making it suitable for use in the manufacturing process of sodium iodide 131I and application in nuclear medicine services.

Automated radiolabelling of [68Ga]Ga-PSMA-11 (gallium (68Ga)-gozetotide) using the Locametz® kit and two generators

BackgroundSteps have been taken by pharmaceutical companies to obtain marketing authorisation of PSMA ligands in the European Union. Since December 2022, Locametz® (PSMA-11, gozetotide) is licensed as kit for manual radiolabelling with gallium-68 and commercially available since mid-2023. The Summary of Product Characteristic (SmPC) describes manual radiolabelling with a maximum activity after radiolabelling of 1369 MBq. We aimed for radiolabelling with a higher activity to increase production efficiency, and thus, automated radiolabelling is strongly preferred over manual radiolabelling to reduce radiation exposure to personnel. The aim of this study was to develop and validate a method for automated radiolabelling of the Locametz® kit using ~ 2000 MBq of gallium-68 eluate for radiolabelling.ResultsAutomated radiolabelling of [68Ga]Ga-PSMA-11 using the Locametz® kit provided a product which complies to the Ph. Eur., had a shelf-life of 6 h at room temperature, and theoretically reduced radiation exposure 5.7 times. Radiolabelling with one and two generator(s) resulted in a radiochemical yield of 91–102% and 96–101% after preparation, respectively. The radiochemical purity ranged from 98.0 to 99.6% for radiolabelling with one generator and ranged from 98.4 to 99.3% for radiolabelling with two generators with similar stability. The activity of the final product was much higher when using two generators, 1961–2035 MBq compared to 740–1260 MBq, which leads to ~ 1.5 times more patient syringes available per preparation.ConclusionAutomated radiolabelling of [68Ga]Ga-PSMA-11 using the Locametz® kit with higher gallium-68 activity than specified in the SmPC results in a product that is in compliance with the Ph. Eur. monograph and has a shelf-life of 6 h at room temperature. Radiolabelling with two generators proved possible and resulted in a product with similar quality but with much higher efficiency.

Production of [211At]NaAt solution under GMP compliance for investigator-initiated clinical trial.

The alpha emitter astatine-211 (211At) is garnering attention as a novel targeted alpha therapy for patients with refractory thyroid cancer resistant to conventional therapy using beta emitter radioiodine (131I). Herein, we aimed to establish a robust method for the manufacturing and quality control of [211At]NaAt solution for intravenous administration under the good manufacturing practice guidelines for investigational products to conduct an investigator-initiated clinical trial. 211At was separated and purified via dry distillation using irradiated Bi plates containing 211At obtained by the nuclear reaction of 209Bi(4He, 2n)211At. After purification, the 211At trapped in the cold trap was collected in a reaction vessel using 15 mL recovery solution (1% ascorbic acid and 2.3% sodium hydrogen carbonate). After stirring the 211At solution for 1h inside a closed system, the reaction solution was passed through a sterile 0.22μm filter placed in a Grade A controlled area and collected in a product vial to prepare the [211At]NaAt solution. According to the 3-lot tests, decay collected radioactivity and radiochemical yield of [211At]NaAt were 78.8 ± 6.0 MBq and 40 ± 3%, respectively. The radiochemical purity of [211At]At- obtained via ion-pair chromatography at the end of synthesis (EOS) was 97 ± 1%, and remained > 96% 6h after EOS; it was detected at a retention time (RT) 3.2-3.3min + RT of I-. LC-MS analysis indicated that this principal peak corresponded with an astatide ion (m/z = 210.988046). In gamma-ray spectrometry, the 211At-related peaks were identified (X-ray: 76.9, 79.3, 89.3, 89.8, and 92.3keV; γ-ray: 569.7 and 687.0keV), whereas the peak at 245.31keV derived from 210At was not detected during the 22h continuous measurement. The target material, Bi, was below the 9 ng/mL detection limit in all lots of the finished product. The pH of the [211At]NaAt solution was 7.9-8.6; the concentration of ascorbic acid was 9-10mg/mL. Other quality control tests, including endotoxin and sterility tests, confirmed that the [211At]NaAt solution met all quality standards. We successfully established a stable method of [211At]NaAt solution that can be administered to humans intravenously as an investigational product.

Synthesis, Screening, and Evaluation of Theranostic Molecular CPCR4-Based Probe Targeting CXCR4.

Chemokines and chemokine receptors are indispensable to play a key role in the development of malignant tumors. As one of the most widely expressed chemokine receptors, chemokine (C-X-C motif) receptor 4 (CXCR4) has been a popular research focus. In most tumors, CXCR4 expression is significantly upregulated. Moreover, integrated nuclide diagnosis and therapy targeting CXCR4 show great potential. [68Ga]Ga-pentixafor, a radioligand targeting CXCR4, exhibits a strong affinity for CXCR4 both in vivo and in vitro. However, [177Lu]Lu-pentixather, the therapeutic companion of [68Ga]Ga-pentixafor, requires significant refinement to mitigate its pronounced hepatic biodistribution. The objective of this study was to synthesize theranostic molecular tracers with superior CXCR4 targeting functions. The Daudi cell line, which highly expressed CXCR4, and the MM.1S cell line, which weakly expressed CXCR4, were used in this study. Based on the pharmacophore cyclo (-d-Tyr-n-me-d-Orn-l-Arg-L-2-NAL-Gly-) (CPCR4) of pentixafor, six tracers were synthesized: [124I]I-1 ([124I]I-CPCR4), [99mTc]Tc-2 ([99mTc]Tc-HYNIC-CPCR4), [124I]I-3 ([124I]I-pentixafor), [18F]AlF-4 ([18F]AlF-NETA-CPCR4), [99mTc]Tc-5 ([99mTc]Tc-MAG3-CPCR4) and [124I]I-6 ([124I]I-pentixafor-Ga) and their radiochemical purities were all higher than 95%. After positron emission tomography (PET)/single-photon emission computed tomography (SPECT) imaging, the [124I]I-6 group exhibited the best target-nontarget ratio. At the same time, comparing the [68Ga]Ga-pentixafor group with the [124I]I-6 group, we found that the [124I]I-6 group had a better target-nontarget ratio and lower uptake in nontarget organs. Therefore, compound 6 was selected for therapeutic radionuclide (131I) labeling, and the tumor-bearing animal models were treated with [131I]I-6. The volume of the tumor site was significantly reduced in the treatment group compared with the control group, and no significant side effects were found. [124I]I-6 and [131I]I-6 showed excellent affinity for targeting CXCR4, and they showed great potential for the integrated diagnosis and treatment of tumors with high CXCR4 expression.

68Ga-DOTA-D-Alanine-BoroPro Radiotracer for Imaging of the Fibroblast Activation Protein in Malignant and Non-Malignant Diseases.

Recently, we reported a new fibroblast activation protein (FAP) inhibitor radiopharmaceutical based on the 99mTc-((R)-1-((6-hydrazinylnicotinoyl)-D-alanyl) pyrrolidin-2-yl) boronic acid (99mTc-HYNIC-D-Alanine-BoroPro)(99mTc-HYNIC-iFAP) structure for tumor microenvironment SPECT imaging. This research aimed to synthesize 68Ga-[2,2',2″,2‴-(2-(4-(2-(5-(((S)-1-((S)-2-boronopyrrolidin-1-yl)-1-oxopropan-2-yl)carbamoyl)pyridin-2-yl)hydrazine-1-carbothioamido)benzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid] (68Ga-DOTA-D-Alanine-BoroPro)(68Ga-iFAP) as a novel radiotracer for PET imaging and evaluate its usefulness for FAP expression in malignant and non-malignant tissues. The coupling of p-SCN-benzene DOTA with HYNIC-iFAP was used for the chemical synthesis and further labeling with 68Ga. Radiochemical purity was verified by radio-HPLC. The specificity of 68Ga-iFAP was evaluated in HCT116 cells, in which FAP expression was verified by immunofluorescence and Western blot. Biodistribution and biokinetic studies were performed in murine models. 68Ga-iFAP uptake at the myocardial level was assessed in mice with induced infarction. First-in-human images of 68Ga-iFAP in healthy subjects and patients with myocardial infarction, glioblastoma, prostate cancer, and breast cancer were also obtained. DOTA-D-Alanine BoroPro was prepared with a chemical purity of 98% and was characterized by UPLC mass spectroscopy, FT-IR, and UV-vis. The 68Ga-iFAP was obtained with a radiochemical purity of >95%. In vitro and in vivo studies demonstrated 68Ga-iFAP-specific recognition for FAP, rapid renal elimination, and adequate visualization of the glioblastoma, breast tumor, prostate cancer, and myocardial infarction sites. The results of this research justify further dosimetry and clinical trials to establish the specificity and sensitivity of 68Ga-iFAP PET for FAP expression imaging.

Legumain-Triggered Macrocyclization of Radiofluorinated Tracer for Enhanced PET Imaging.

Enhancing the accumulation and retention of small-molecule probes in tumors is an important way to achieve accurate cancer diagnosis and therapy. Enzyme-stimulated macrocyclization of small molecules possesses great potential for enhanced positron emission tomography (PET) imaging of tumors. Herein, we reported an 18F-labeled radiotracer [18F]AlF-RSM for legumain detection in vivo. The tracer was prepared by a one-step aluminum-fluoride-restrained complexing agent ([18F]AlF-RESCA) method with high radiochemical yield (RCY) (88.35 ± 3.93%) and radiochemical purity (RCP) (>95%). More notably, the tracer can be transformed into a hydrophobic macrocyclic molecule under the joint action of legumain and reductant. Simultaneously, the tracer could target legumain-positive tumors and enhance accumulation and retention in tumors, resulting in the amplification of PET imaging signals. The enhancement of radioactivity enables PET imaging of legumain activity with high specificity. We envision that, by combining this highly efficient 18F-labeled strategy with our intramolecular macrocyclization reaction, a range of radiofluorinated tracers can be designed for tumor PET imaging and early cancer diagnosis in the future.