EANM guideline on quality risk management for radiopharmaceuticals.

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

Theragnostic radionuclides: a clinical perspective.

The concept of theragnostics goes back to the earliest days of nuclear medicine, with [123I/131I]iodide in thyroid disease and [123I/131I]MIBG in phaeochromocytoma being examples in long-term use. However, in recent years there has been a great expansion in the application of theragnostics, beginning with [68Ga/177Lu]-labelled somatostatin peptides for evaluation and treatment of neuroendocrine tumors. We are currently seeing the rapid development of [68Ga/177Lu]PSMA theragnostics in metastatic prostate cancer. While these applications are very promising, there are a number of practicalities which must be addressed in the development and introduction of novel theragnostics. The physical half-lives of the diagnostic and therapeutic radionuclides must be appropriate for imaging and delivery of targeted cell killing, respectively. The types of radioactive emissions are critical; beta particles can traverse several millimeters but also risk damaging non-target tissues, while alpha particles deliver their energy over a much shorter path length, a few cell diameters, and must be more directly targeted. It must be practical to produce the therapeutic radionuclide and the final radiopharmaceutical and deliver them to the final user within an appropriate time-frame determined by half-life and stability. The biodistribution of the agent must demonstrate adequate accumulation and retention in the target tissue with clearance from adjacent and/or radio-sensitive normal tissues. The commercial success of recently introduced theragnostics suggests a rosy future for personalized medicine.

Guidelines on nuclear medicine imaging in neuroblastoma.

Nuclear medicine has a central role in the diagnosis, staging, response assessment and long-term follow-up of neuroblastoma, the most common solid extracranial tumour in children. These EANM guidelines include updated information on 123I-mIBG, the most common study in nuclear medicine for the evaluation of neuroblastoma, and on PET/CT imaging with 18F-FDG, 18F-DOPA and 68Ga-DOTA peptides. These PET/CT studies are increasingly employed in clinical practice. Indications, advantages and limitations are presented along with recommendations on study protocols, interpretation of findings and reporting results.

Shining dead bone-cause for cautious interpretation of [18F]NaF PET scans.

Background and purpose - [18F]Fluoride ([18F]NaF) PET scan is frequently used for estimation of bone healing rate and extent in cases of bone allografting and fracture healing. Some authors claim that [18F]NaF uptake is a measure of osteoblastic activity, calcium metabolism, or bone turnover. Based on the known affinity of fluoride to hydroxyapatite, we challenged this view. Methods - 10 male rats received crushed, frozen allogeneic cortical bone fragments in a pouch in the abdominal wall on the right side, and hydroxyapatite granules on left side. [18F]NaF was injected intravenously after 7 days. 60 minutes later, the rats were killed and [18F]NaF uptake was visualized in a PET/CT scanner. Specimens were retrieved for micro CT and histology. Results - MicroCT and histology showed no signs of new bone at the implant sites. Still, the implants showed a very high [18F]NaF uptake, on a par with the most actively growing and remodeling sites around the knee joint. Interpretation - [18F]NaF binds with high affinity to dead bone and calcium phosphate materials. Hence, an [18F]NaF PET/CT scan does not allow for sound conclusions about new bone ingrowth into bone allograft, healing activity in long bone shaft fractures with necrotic fragments, or remodeling around calcium phosphate coated prostheses.

Radiosynthesis of the iodine-124 labeled Hsp90 inhibitor PU-H71.

Heat shock protein 90 (Hsp90) is an ATP dependent molecular chaperone protein whose function is critical for maintaining several key proteins involved in survival and proliferation of cancer cells. PU-H71 (1), is a potent purine-scaffold based ATP pocket binding Hsp90 inhibitor which has been shown to have potent activity in a broad range of in vivo cancer models and is currently in Phase I clinical trials in patients with advanced solid malignancies, lymphomas, and myeloproliferative neoplasms. In this report, we describe the radiosynthesis of [(124)I]-PU-H71(5); this was synthesized from the corresponding Boc-protected stannane precursor 3 by iododestannylation with [(124)I]-NaI using chloramine-T as an oxidant for 2 min, followed by Boc deprotection with 6 N HCl at 50 °C for 30 min to yield the final compound. The final product 5 was purified using HPLC and was isolated with an overall yield of 55 ± 6% (n = 6, isolated) from 3, and >98% purity and an average specific activity of 980 mCi/µmol. Our report sets the stage for the introduction of [(124)I]-PU-H71 as a potential non-invasive probe for understanding biodistribution and pharmacokinetics of PU-H71 in living subjects using positron emission tomography imaging.

EANM guideline for the preparation of an Investigational Medicinal Product Dossier (IMPD).

The preparation of an Investigational Medicinal Product Dossier (IMPD) for a radiopharmaceutical to be used in a clinical trial is a challenging proposition for radiopharmaceutical scientists working in small-scale radiopharmacies. In addition to the vast quantity of information to be assembled, the structure of a standard IMPD is not well suited to the special characteristics of radiopharmaceuticals. This guideline aims to take radiopharmaceutical scientists through the practicalities of preparing an IMPD, in particular giving advice where the standard format is not suitable. Examples of generic IMPDs for three classes of radiopharmaceuticals are given: a small molecule, a kit-based diagnostic test and a therapeutic radiopharmaceutical.

Guidance on current good radiopharmacy practice for the small-scale preparation of radiopharmaceuticals using automated modules: a European perspective.

This document is meant to complement Part B of the EANM 'Guidelines on current good radiopharmacy practice (cGRPP) in the preparation of radiopharmaceuticals' issued by the Radiopharmacy Committee of the European Association of Nuclear Medicine, covering small-scale in-house preparation of radiopharmaceuticals with automated modules. The aim is to provide more detailed and practice-oriented guidance to those who are involved in the small-scale preparation of radiopharmaceuticals, which are not intended for commercial purposes or distribution.

Guideline on current good radiopharmacy practice (cGRPP) for the small-scale preparation of radiopharmaceuticals.

This guideline on current good radiopharmacy practice (cGRPP) for small-scale preparation of radiopharmaceuticals represents the view of the Radiopharmacy Committee of the European Association of Nuclear Medicine (EANM). The guideline is laid out in the format of the EU Good Manufacturing Practice (GMP) guidelines as defined in EudraLex volume 4. It is intended for non-commercial sites such as hospital radiopharmacies, nuclear medicine departments, research PET centres and in general any healthcare establishments. In the first section, general aspects which are applicable to all levels of operations are discussed. The second section discusses the preparation of small-scale radiopharmaceuticals (SSRP) using licensed generators and kits. Finally, the third section goes into the more complex preparation of SSRP from non-licensed starting materials, often requiring a purification step and sterile filtration. The intention is that the guideline will assist radiopharmacies in the preparation of diagnostic and therapeutic SSRP's safe for human administration.

EANM guideline on the validation of analytical methods for radiopharmaceuticals.

BACKGROUND: To fulfil good manufacturing requirements, analytical methods for the analysis of pharmaceuticals for human and vetinary use must be validated. The International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) has published guidance documents on the requirements for such validation activities and these have been adopted by the European Medicines Agency, The U.S. Food and Drug Administration (FDA) and other regulatory bodies. These guidance documents do not, however, fully address all the specific tests required for the analysis of radiopharmaceuticals. This guideline attempts to rectify this shortcoming, by recommending approaches to validate such methods. RESULTS: Recommedations for the validation of analytical methods which are specific for radiopharmaceutials are presented in this guideline, along with two practical examples. CONCLUSIONS: In order to comply with good manufacturing practice, analytical methods for radiopharmaceuticals for human use should be validated.

Paradigms for Precision Medicine in Epichaperome Cancer Therapy.

Alterations in protein-protein interaction networks are at the core of malignant transformation but have yet to be translated into appropriate diagnostic tools. We make use of the kinetic selectivity properties of an imaging probe to visualize and measure the epichaperome, a pathologic protein-protein interaction network. We are able to assay and image epichaperome networks in cancer and their engagement by inhibitor in patients' tumors at single-lesion resolution in real time, and demonstrate that quantitative evaluation at the level of individual tumors can be used to optimize dose and schedule selection. We thus provide preclinical and clinical evidence in the use of this theranostic platform for precision medicine targeting of the aberrant properties of protein networks.

In vivo 19F magnetic resonance spectroscopy and chemical shift imaging of tri-fluoro-nitroimidazole as a potential hypoxia reporter in solid tumors.

PURPOSE: 2-Nitro-alpha-[(2,2,2-trifluoroethoxy)methyl]-imidazole-1-ethanol (TF-MISO) was investigated as a potential noninvasive marker of tissue oxygen levels in tumors using (19)F magnetic resonance spectroscopy (MRS) and (19)F chemical shift imaging. EXPERIMENTAL DESIGNS: In vitro data were obtained using high-performance liquid chromatography on tumor cells incubated under varying oxygen conditions to determine the oxygen-binding characteristics. In vivo data were obtained using a well-characterized hypoxic murine breast tumor (MCa), in addition to studies on a rat prostate tumor model (R3327-AT) implanted in nude mice. Detection of intratumor (19)F signal from TF-MISO was done using MRS for up to 10 h following a 75 mg/kg i.v. injection. Localized distribution of the compound in the implanted MCa tumor has been imaged using slice-selective two-dimensional chemical shift imaging 6 h after injection. RESULTS: The in vitro results showed that TF-MISO preferentially accumulates in cells incubated under anoxic conditions. The in vivo (19)F MR spectral features (line width and chemical shift) were recorded as a function of time after injection, and the results indicate that the fluorine atoms are indeed sensitive to changes in the local environment while still providing a detectable MR signal. Ex vivo spectra were collected and established the visibility of the (19)F signal under conditions of maximum hypoxia. Late time point (>6 h) tumor tissue concentrations, as obtained from (19)F MRS, suggest that TF-MISO is reduced and retained in hypoxic tumor. The feasibility of obtaining TF-MISO tumor distribution maps in a reasonable time frame was established. CONCLUSIONS: Based on the results presented herein, it is suggested that TF-MISO has the potential to be a valid magnetic resonance hypoxia imaging reporter for both preclinical hypoxia studies and hypoxia-directed clinical therapy.

Radiolabeled Nanoparticles for Cancer Diagnosis and Therapy.

Cancer remains as one of the major causes of death worldwide. The emergence of nanotechnology has opened new avenues for the development of nanoparticle (NP)- based diagnostic and therapeutic tools. NPs of different chemical composition, size, shape and surface decoration can be prepared using a wide variety of synthetic strategies. Subsequent radiolabelling with positron or gamma emitters results in potential diagnostic agents which may offer improved selectivity and/or specificity for the target organ or tissue, enabling the acquisition of images with higher signal-to-contrast ratio. Incorporation of alpha or beta emitters leads to therapeutic agents with application in the field of radiotherapy. Here, we first describe the different labeling strategies reported so far for the incorporation of radionuclides into NPs. Recent advances in the use of nanoparticulate constructs both in the diagnostic and therapeutic arenas are then discussed and examples of their application are briefly discussed.

A two-center study for the quality control of [(18)F]FDG using FASTlab phosphate cassettes.

OBJECTIVE: The GE FASTlab radiosynthesis module is routinely used for the production of [(18)F]FDG, utilizing the commercially available phosphate cassettes. Because of the observation of a white precipitate in the product vial before the product expiry time, we re-examined the quality of the produced [(18)F]FDG solution. METHODS: Phosphate buffered [(18)F]FDG solution was synthesized on the FASTlab and analyzed at both National Taiwan University Hospital (NTUH) of Taiwan and Royal Brisbane and Women's Hospital (RBWH) of Australia. In addition to the standard product quality control (QC), the concentration of aluminum (Al(3+)) as probable cause of the precipitations in the [(18)F]FDG solution was analyzed by inductively coupled plasma mass spectrometry (ICP-MS at RBWH) and inductively coupled plasma optical emission spectrometry (ICP-OES at NTUH), and using three semi-quantitative methods at NTUH, Advantec(®) Alumi Check Test Strip, Quantofix(®) Aluminum Test Strip and MColortest™ Aluminum Test kit. RESULTS: The precipitates were observed in the [(18)F]FDG solution within 24 (NTUH) and 6 (RBWH) hours after the end of synthesis in 38-100 % of the batches, dependent on the batch of the FASTlab cassettes. Addition of metal-free HCl(aq) to aliquots of [(18)F]FDG containing precipitate, followed by ICP-MS analysis revealed Al(3+) concentrations of 70-80 ppm. Al(3+) concentrations of 10-12 ppm were detected in [(18)F]FDG batches that did not show any precipitation. In contrast, less than 5 ppm of the residual Al(3+) was detected by semi-quantitative methods in all batches. CONCLUSION: The US (USP), British (BP), European (EP) and Japanese (JP) pharmacopeias demand that [(18)F]FDG for injection should be clear and particulate free within the given shelf-life/expiration time. To avoid Al-phosphate precipitation within the product expiry time, FASTlab citrate cassettes, rather than phosphate cassettes, should be used for [(18)F]FDG production. Although testing for Al(3+) is not listed in the [(18)F]FDG monographs of the USP, BP and EP, residual Al(3+) levels should be considered in the interests of patient safety.

Synthesis of radiolabelled aryl azides from diazonium salts: experimental and computational results permit the identification of the preferred mechanism.

Experimental and computational studies on the formation of aryl azides from the corresponding diazonium salts support a stepwise mechanism via acyclic zwitterionic intermediates. The low energy barriers associated with both transition structures are compatible with very fast and efficient processes, thus making this method suitable for the chemical synthesis of radiolabelled aryl azides.

Iodination of annexin V for imaging apoptosis.

UNLABELLED: Our goal in this investigation was to develop a method for iodinating annexin V that would be suitable for the in vivo detection of apoptosis. METHODS: Annexin V was iodinated with (125)I using 2 different techniques: direct iodination with IODO-BEADS, resulting in the iodination of tyrosine residues; and use of the Bolton-Hunter reagent, which binds to lysine. The active fraction of the labeled preparation was purified by affinity chromatography. We assessed thyroid accumulation of free iodide by comparing mice with blocked and unblocked thyroids. We tested the ability of iodinated annexin V to bind apoptotic cells in vitro using irradiated neuroblastoma cells and immobilized phosphatidylserine and in vivo using C3H mice subjected to whole-body irradiation. RESULTS: The efficiency of IODO-BEADS iodination was just below 30%; with the Bolton-Hunter protocol we were able to achieve 40% efficiency. When the IODO-BEADS-labeled preparation was injected into nude mice, activity accumulated rapidly in the thyroid. Two hours after injection, uptake in the thyroid region was clearly visible on a gamma-camera scan. This uptake was absent in mice that had had their thyroids blocked. We concluded that the IODO-BEADS method of labeling resulted in a protein that was rapidly deiodinated in vivo. By contrast, when annexin V was labeled using the Bolton-Hunter protocol, there was no evidence of activity accumulating in the thyroid. The Bolton-Hunter-labeled annexin V bound to apoptotic cells and immobilized phosphatidylserine in vitro. The active fraction of Bolton-Hunter-labeled annexin V was approximately 0.75. In C3H mice given 5-Gy whole-body irradiation, there was a significant induction of apoptosis in the spleen, as measured by the terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling assay, and a 4-fold increase in (125)I activity in the spleens relative to that of the control animals. CONCLUSION: Direct iodination of annexin V on tyrosine residues is a poor technique suffering from rapid deiodination in vivo. With Bolton-Hunter chemistry, one can produce a molecule that retains its label in vivo and binds to apoptotic cells in vitro and in vivo.

Influence of free fatty acids on glucose uptake in prostate cancer cells.

INTRODUCTION: The study focuses on the interaction between glucose and free fatty acids (FFA) in malignant human prostate cancer cell lines by an in vitro observation of uptake of fluoro-2-deoxy-D-glucose (FDG) and acetate. METHODS: Human prostate cancer cell lines (PC3, CWR22Rv1, LNCaP, and DU145) were incubated for 2 h and 24 h in glucose-containing (5.5 mM) Dulbecco's Modified Eagle's Medium (DMEM) with varying concentrations of the free fatty acid palmitate (0-1.0 mM). Then the cells were incubated with [(18)F]-FDG (1 µCi/mL; 0.037 MBq/mL) in DMEM either in presence or absence of glucose and in presence of varying concentrations of palmitate for 1 h. Standardized procedures regarding cell counting and measuring for (18)F radioactivity were applied. Cell uptake studies with (14)C-1-acetate under the same conditions were performed on PC3 cells. RESULTS: In glucose containing media there was significantly increased FDG uptake after 24 h incubation in all cell lines, except DU145, when upper physiological levels of palmitate were added. A 4-fold increase of FDG uptake in PC3 cells (15.11% vs. 3.94%/10(6) cells) was observed in media with 1.0 mM palmitate compared to media with no palmitate. The same tendency was observed in PC3 and CWR22Rv1 cells after 2 h incubation. In glucose-free media no significant differences in FDG uptake after 24 h incubation were observed. The significant differences after 2 h incubation all pointed in the direction of increased FDG uptake when palmitate was added. Acetate uptake in PC3 cells was significantly lower when palmitate was added in glucose-free DMEM. No clear tendency when comparing FDG or acetate uptake in the same media at different time points of incubation was observed. CONCLUSIONS: Our results indicate a FFA dependent metabolic boost/switch of glucose uptake in PCa, with patterns reflecting the true heterogeneity of the disease.

Antilipolytic drug boosts glucose metabolism in prostate cancer.

INTRODUCTION: The antilipolytic drug Acipimox reduces free fatty acid (FFA) levels in the blood stream. We examined the effect of reduced FFAs on glucose metabolism in androgen-dependent (CWR22Rv1) and androgen-independent (PC3) prostate cancer (PCa) xenografts. METHODS: Subcutaneous tumors were produced in nude mice by injection of PC3 and CWR22Rv1 PCa cells. The mice were divided into two groups (Acipimox vs. controls). Acipimox (50mg/kg) was administered by oral gavage 1h before injection of tracers. 1h after i.v. co-injection of 8.2MBq (222 ± 6.0 µCi) (18)F-FDG and~0.0037 MBq (0.1 µCi) (14)C-acetate, (18)F-FDG imaging was performed using a small-animal PET scanner. Counting rates in reconstructed images were converted to activity concentrations. Quantification was obtained by region-of-interest analysis using dedicated software. The mice were euthanized, and blood samples and organs were harvested. (18)F radioactivity was measured in a calibrated γ-counter using a dynamic counting window and decay correction. (14)C radioactivity was determined by liquid scintillation counting using external standard quench corrections. Counts were converted into activity, and percentage of the injected dose per gram (%ID/g) tissue was calculated. RESULTS: FDG biodistribution data in mice with PC3 xenografts demonstrated doubled average %ID/g tumor tissue after administration of Acipimox compared to controls (7.21 ± 1.93 vs. 3.59 ± 1.35, P=0.02). Tumor-to-organ ratios were generally higher in mice treated with Acipimox. This was supported by PET imaging data, both semi-quantitatively (mean tumor FDG uptake) and visually (tumor-to-background ratios). In mice with CWR22Rv1 xenografts there was no effect of Acipimox on FDG uptake, either in biodistribution or PET imaging. (14)C-acetate uptake was unaffected in PC3 and CWR22Rv1 xenografts. CONCLUSIONS: In mice with PC3 PCa xenografts, acute administration of Acipimox increases tumor uptake of (18)F-FDG with general improvements in tumor-to-background ratios. Data indicate that administration of Acipimox prior to (18)F-FDG PET scans has potential to improve sensitivity and specificity in patients with castration-resistant advanced PCa.

68Ga-autoclabeling of DOTA-TATE and DOTA-NOC.

INTRODUCTION: A new method combining (68)Ga-labeling and steam sterilization, here called autoclabeling, has been evaluated for two somatostatin receptor binding tracers used for positron emission tomography (PET) imaging of neuroendocrine tumors; DOTA-TATE and -NOC. METHODS: The two peptides DOTA-TATE and -NOC were labeled with (68)Ga by heating for 15 min at 121°C in the presence of acetate buffer at pH 4.3. The product solutions were tested for sterility, presence of endotoxins, degradation of peptide and osmolality. RESULTS: Complete incorporation of (68)Ga was obtained after the autoclabeling reaction and no degradation of the peptides was observed. Sterility was verified and the presence of endotoxins was well within Ph. Eur limits (175IU/maximum injected volume). CONCLUSIONS: The autoclabeling method provides a convenient procedure for (68)Ga-labeling by combining the labeling reaction and steam sterilization into one single step.

A quartz-lined carbon-11 target: striving for increased yield and specific activity.

INTRODUCTION: The increased demand for high specific radioactivity neuroreceptor ligands for positron emission tomography (PET) requires the production of high specific radioactivity carbon-11 in high yields. We have attempted to address this issue with the development of a new quartz-lined aluminium target for the production of [(11)C]methane or [(11)C]carbon dioxide. METHODS: The new target has been tested with respect to yields of [(11)C]methane and [(11)C]carbon dioxide, and the effect of the quartz liner has been evaluated. The specific radioactivities of a large number of radiopharmaceuticals produced using this target have also been measured. RESULTS: The described target produces [(11)C]-labelled gases in excellent yields, and losses of radioactivity in the target on production of [(11)C]methane have been reduced significantly by the use of a quartz liner. Radiopharmaceuticals with specific radioactivities up to 9000 GBq/µmol at end of bombardment (EOB) (243 Ci/µmol) have been produced using this target. CONCLUSIONS: We have developed a reliable, high-yielding carbon-11 gas target which is now routinely used in our department for the production of high specific activity radiopharmaceuticals.