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.

Current Status of Radiopharmaceuticals for the Theranostics of Neuroendocrine Neoplasms.

Nuclear medicine plays a pivotal role in the management of patients affected by neuroendocrine neoplasms (NENs). Radiolabeled somatostatin receptor analogs are by far the most advanced radiopharmaceuticals for diagnosis and therapy (radiotheranostics) of NENs. Their clinical success emerged receptor-targeted radiolabeled peptides as an important class of radiopharmaceuticals and it paved the way for the investigation of other radioligand-receptor systems. Besides the somatostatin receptors (sstr), other receptors have also been linked to NENs and quite a number of potential radiolabeled peptides have been derived from them. The Glucagon-Like Peptide-1 Receptor (GLP-1R) is highly expressed in benign insulinomas, the Cholecystokinin 2 (CCK2)/Gastrin receptor is expressed in different NENs, in particular medullary thyroid cancer, and the Glucose-dependent Insulinotropic Polypeptide (GIP) receptor was found to be expressed in gastrointestinal and bronchial NENs, where interestingly, it is present in most of the sstr-negative and GLP-1R-negative NENs. Also in the field of sstr targeting new discoveries brought into light an alternative approach with the use of radiolabeled somatostatin receptor antagonists, instead of the clinically used agonists. The purpose of this review is to present the current status and the most innovative strategies for the diagnosis and treatment (theranostics) of neuroendocrine neoplasms using a cadre of radiolabeled regulatory peptides targeting their receptors.

Imaging and 1-day kinetics of intracoronary stem cell transplantation in patients with idiopathic dilated cardiomyopathy.

BACKGROUND: Stem cell transplantation is an emerging method of treatment for patients with cardiovascular disease. There are few studies completed or ongoing on stem cell therapy in patients with idiopathic dilated cardiomyopathy (IDCM). Information on stem cell homing and distribution in the myocardium after transplantation might provide important insight into effectiveness of transplantation procedure. AIM: To assess early engraftment, retention and migration of intracoronarily transplanted stem cells in the myocardium of patients with advanced dilated cardiomyopathy of non-ischaemic origin using stem cell labeling with (99m)Tc-exametazime (HMPAO). MATERIALS, METHODS: Thirty-five patients with IDCM and advanced heart failure were included in the study. Autologous hematopoietic (CD34+) stem cells were harvested by peripheral blood apheresis after bone marrow stimulation, labeled with (99m)Tc-HMPAO, tested for viability and injected into coronary vessel supplying areas of myocardium selected by myocardial perfusion scintigraphy as dysfunctional yet viable. Imaging was performed 1h and 18h after transplantation. RESULTS: Myocardial stem cell retention ranged from 0 to 1.44% on early and 0-0.97% on delayed imaging. Significant efflux of stem cells occurred from site of delivery in this time period (p<0.001). Stem cell viability was not affected by labeling. CONCLUSION: Stem cell labeling with (99m)Tc-HMPAO is a feasible method for stem cell tracking after transplantation in patients with IDCM.

Intracoronary transplantation of CD34(+) cells is associated with improved myocardial perfusion in patients with nonischemic dilated cardiomyopathy.

BACKGROUND: We investigated the effects of intracoronary transplantation of CD34(+) cells on myocardial perfusion in patients with nonischemic dilated cardiomyopathy (DCM). METHODS AND RESULTS: We enrolled 21 patients with DCM (left ventricular ejection fraction [LVEF] <40%, New York Heart Association functional class III) who underwent peripheral stem cell mobilization with granulocyte-colony stimulating factor (G-CSF). CD34(+) cells were collected by means of apheresis. Patients underwent myocardial perfusion imaging, and CD34(+) cells were injected in the coronary artery supplying viable segments with reduced myocardial perfusion and regional dysfunction. Myocardial perfusion imaging was repeated 6 months later. Clinical response to stem cell therapy was predefined as a change in LVEF >5%. The majority of patients were men (81%) with an overall mean age 53 ± 9 years, LVEF 25 ± 5%, and 6-minute walking distance 354 ± 71 m. Myocardial perfusion defects at rest were observed in 86% of patients and were more common in the left anterior descending territory (50%). At 6 months' follow-up, there was a significant improvement in rest myocardial perfusion scores (6.3 ± 5.8 vs 3.1 ± 4.3; P < .001), LVEF (25 ± 7% vs 29 ± 8%; P = .005), and 6-minute walking distance (354 ± 71 m vs 404 ± 91 m; P < .001). Responders to stem cell therapy had lower summed rest perfusion score at both baseline (3.2 ± 3.0 vs 9.1 ± 6.3; P = .015) and follow-up (1.0 ± 1.5 vs 5.0 ± 5.1; P = .028). CONCLUSIONS: CD34(+) cell transplantation may lead to improved myocardial perfusion in patients with nonischemic DCM. Patients with less severe myocardial perfusion defects at baseline may have an increased likelihood to respond to intracoronary CD34(+) cell transplantation.

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.

Comparative biodistribution of 12 ¹¹¹In-labelled gastrin/CCK2 receptor-targeting peptides.

PURPOSE: Cholecystokinin 2 (CCK-2) receptor overexpression has been demonstrated in various tumours such as medullary thyroid carcinomas and small-cell lung cancers. Due to this high expression, CCK-2 receptors might be suitable targets for radionuclide imaging and/or radionuclide therapy. Several CCK-2 receptor-binding radiopeptides have been developed and some have been tested in patients. Here we aimed to compare the in vivo tumour targeting properties of 12 (111)In-labelled 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-conjugated gastrin/CCK2 receptor-binding peptides. METHODS: Two CCK8-based peptides and ten gastrin-based peptide analogues were tested. All peptides were conjugated with DOTA and labelled with (111)In. Biodistribution studies were performed in mice with subcutaneous CCK2/gastrin receptor-expressing tumours and with receptor-negative tumours contralaterally. Biodistribution was studied by counting dissected tissues at 1 and 4 h after injection. RESULTS: Both the CCK analogues displayed relatively low tumour uptake (approximately 2.5%ID/g) as compared to minigastrin analogues. Two linear minigastrin peptides (MG0 and sargastrin) displayed moderate tumour uptake at both 1 and 4 h after injection, but also very high kidney uptake (both higher than 48%ID/g). The linear MG11, lacking the penta-Glu sequence, showed lower tumour uptake and also low kidney uptake. Varying the N-terminal Glu residues in the minigastrin analogues led to improved tumour targeting properties, with PP-F11 displaying the optimal biodistribution. Besides the monomeric linear peptides, a cyclized peptide and a divalent peptide were tested. CONCLUSION: Based on these studies, optimal peptides for peptide receptor radionuclide targeting of CCK2/gastrin receptor-expressing tumours were the linear minigastrin analogue with six D-Glu residues (PP-F11), the divalent analogue MGD5 and the cyclic peptide cyclo-MG1. These peptides combined high tumour uptake with low kidney retention, and may therefore be good candidates for future clinical studies.

Comparison of the binding and internalization properties of 12 DOTA-coupled and ¹¹¹In-labelled CCK2/gastrin receptor binding peptides: a collaborative project under COST Action BM0607.

PURPOSE: Specific overexpression of cholecystokinin 2 (CCK2)/gastrin receptors has been demonstrated in several tumours of neuroendocrine origin. In some of these cancer types, such as medullary thyroid cancer (MTC), a sensitive diagnostic modality is still unavailable and therapeutic options for inoperable lesions are needed. Peptide receptor radionuclide therapy (PRRT) may be a viable therapeutic strategy in the management of these patients. Several CCK2R-targeted radiopharmaceuticals have been described in recent years. As part of the European Union COST Action BM0607 we studied the in vitro and in vivo characteristics of 12 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-conjugated CCK2R binding peptides. In the present study, we analysed binding and internalization characteristics. Stability, biodistribution and imaging studies have been performed in parallel by other centres involved in the project. METHODS: Determination of IC(50) values was performed using autoradiography, with DOTA-peptides displacing (125)I-CCK from receptors on tissue sections from human tumours. Saturation binding and internalization experiments were performed using (111)In-labelled peptides. The rat AR42J cell line and the human A431-CCK2R transfected cell line were utilized for in vitro experiments; dissociation constants (K(d)) and apparent number of binding sites (B(max)) were determined. Internalization was determined in receptor-expressing cells by incubating with tracer amounts of peptide at 37 and 4°C for different times up to 120 min. Surface-bound peptide was then stripped either by acid wash or subsequent incubation with 1 µM unlabelled peptide at 4°C. RESULTS: All peptides showed high receptor affinity with IC(50) values ranging from 0.2 to 3.4 nM. Saturation experiments also showed high affinity with K(d) values in the 10(-9)-10(-8) M range. B(max) values estimated in A431-CCK2R cells ranged from 0.6 to 2.2 × 10(6) per cell. All peptides showed high levels of internalization when incubated at 37°C. CONCLUSION: All DOTA-conjugated peptides showed high receptor binding and internalization properties and appear suitable for further characterization, as described in other articles of this issue.

Comparison of biological stability and metabolism of CCK2 receptor targeting peptides, a collaborative project under COST BM0607.

PURPOSE: Stability of radiolabelled cholecystokinin 2 (CCK2) receptor targeting peptides has been a major limitation in the use of such radiopharmaceuticals especially for targeted radionuclide therapy applications, e.g. for treatment of medullary thyroid carcinoma (MTC). The purpose of this study was to compare the in vitro stability of a series of peptides binding to the CCK2 receptor [selected as part of the COST Action on Targeted Radionuclide Therapy (BM0607)] and to identify major cleavage sites. METHODS: Twelve different 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA)-minigastrin/CCK conjugates were provided within an European COST Action (BM0607) by different laboratories and radiolabelled with (177)Lu. Their in vitro stabilities were tested in fresh human serum. Radiochemical yields (RCY) and intact radioligands for half-life calculations were determined by radio-HPLC. Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) analysis of metabolites was performed to identify cleavage products using conjugates labelled with excess stable (nat)Lu, incubated in serum at 37°C. Urine metabolite analysis after injection in normal mice was performed by radio-HPLC analysis. RESULTS: Variable stability in human serum was found for the different peptides with calculated half-lives between 4.5 ± 0.1 h and 198 ± 0.1 h (n = 2). In urine of normal mice only metabolised peptide fragments were detected even at short times after injection for all peptides. MALDI-TOF MS revealed a major cleavage site of all minigastrin derivatives between Asp and Phe-NH(2) at the C-terminal end. CONCLUSION: Development of CCK2 receptor ligands especially for therapeutic purposes in patients with MTC or small cell lung cancer (SCLC) is still ongoing in different laboratories. This comparative study provided valuable insight into the importance of biological stability especially in the context of other results of this comparative trial within the COST Action BM0607.

Influence of radiation source geometry on determination of (111)In and (90)Y activity of radiopharmaceuticals.

OBJECTIVE: Yttrium-90 (Y)-labelled peptides such as DOTATOC and antibodies such as Zevalin are widely used in radionuclide therapy. Indium-111 (In) is used as a Y surrogate for imaging and dosimetry purposes. We aimed to investigate accuracy, geometry (vials and syringes) and volume dependencies for both radionuclides in several different radionuclide calibrators. METHODS: YCl3 and InCl3 solutions were gravimetrically dispensed into the most frequently used containers. In each container several dilutions of the parent solutions were performed. Mass, activity and time were recorded for each calibrator and measurement. Aliquots of both parent solutions were calibrated at the National Metrology Laboratory, Vienna, Austria (BEV). From our measurements and results from BEV, correction factors were determined and further partitioned into calibration, geometry and volume correction factors. RESULTS: Using the nominal calibration factors provided by the manufacturer, measured activity in P6 vials was overestimated by up to 25% for In, depending on the calibrator. Y activity was either underestimated (by up to 20%) or overestimated (by up to 25%) using different radionuclide calibrators. This is the result of the difference in containers used to set the manufacturer's calibration factor values and the containers used in nuclear medicine departments and in this study. There was little geometry dependence for glass vials but strong geometry dependence for syringes for both radionuclides in all calibrators. CONCLUSION: The results should constitute a warning for all personnel responsible for preparation of radiopharmaceuticals. Every nuclear medicine department should incorporate a proper quality-control regimen for radionuclide calibrators and a quality-assurance system.

99mTc-labelled rituximab, a new non-Hodgkin's lymphoma imaging agent: first clinical experience.

OBJECTIVE: This study was performed to explore the possibility of using Tc-rituximab as an imaging agent to assess expression of CD20 antigen in patients with B-cell non-Hodgkin's lymphoma (NHL) before (radio) immunotherapy, for staging and subsequent evaluation of remission of NHL. METHODS: Rituximab was purified from Mabthera and photoactivated by ultraviolet light. The irradiated solution was aliquoted and labelled with pertechnetate. The effectiveness of the labelling method was evaluated by determination of the number of free thiol groups per photoreduced antibody, radiochemical purity determination and in-vitro stability. Immunoreactivity of Tc-rituximab was assessed on Ramos cells using a direct binding assay. Ten patients (age 31-70 years, mean 50 years) were included, nine with CD20 B-cell NHL and one with CD20-NHL. Whole-body and single photon emission computed tomography images were taken 1, 3, 6 and 20 h postinjection of Tc-rituximab. Scintigraphic results were compared with computerized tomography (CT) findings. RESULTS: In all cases radiochemical purity over 95% was observed with preserved affinity for CD20 antigen. In all patients expected activity was seen in the blood pool, liver, kidneys and spleen. Pathological, moderately to markedly increased Tc-rituximab activity was seen in all but one CT-confirmed NHL involved sites 6 and 20 h postinjection. In one patient, increased activity of Tc-rituximab was additionally seen in one region not seen on CT. In three patients increased accumulation was seen in bone marrow. CONCLUSION: Tc-rituximab is a promising imaging agent suitable for assessing expression of CD20 in patients with NHL before (radio) immunotherapy.

Adsorption of radiopharmaceuticals to syringes: setting up a reliable protocol for its assessment.

BACKGROUND: It is well known that various drugs, including radiopharmaceuticals, may adsorb to plastic syringes to different extents. Some reports suggest that adsorption can reach levels of almost 50%. The consequence of adsorption of a radiopharmaceutical and subsequent inadequate dosing can include an inappropriate diagnostic response in patients, an increase in the duration of investigation or treatment, and an increase in cost. AIM: To investigate the extent of adsorption of Tc-succimer to plastic syringes and to set up a reliable protocol for assessment of the extent of retention of drugs to single-use plastic syringes before a new syringe brand and/or radiopharmaceutical are introduced in a nuclear medicine department. METHODS: Radiopharmaceutical kits from two different manufacturers were compared for retention using syringes from three different producers. To assess the influence of dilution on retention, Tc-succimer preparations were further diluted with sodium chloride solution for injection. Syringes were filled to one-third of their capacity and incubated at room temperature before being emptied into vacuum vials. The radioactivity of the syringes was measured before and after emptying. The extent of retained radioactivity was calculated as a percentage of radioactivity in the syringe before emptying. RESULTS: Results show that adsorption of Tc-succimer considerably differs between syringe brands. The adsorption of undiluted Tc-succimer in 30 min may exceed 50%. CONCLUSION: The findings show that measurement of retention in syringes and injection sets should be regarded as an essential aspect of quality assurance before radiopharmaceuticals, syringes and injection sets are used routinely.