On-cartridge preparation and evaluation of 68Ga-, 89Zr- and 64Cu-precursors for cell radiolabelling.

INTRODUCTION: Indium-111 when formulated as indium-111 oxine remains the gold standard for long term cell tracking, whereas radiometals for improved PET applications still have to be established. We here describe the on-cartridge formation of gallium-68, zirconium-89 and copper-64 complexes in small volumes suitable for cell labelling, including labelling of red blood cells (RBC) and white blood cells (WBC) and their biological evaluation in vivo. METHODS: Small volumes (1-2 mL) of tracers (oxine, tropolone) were directly prepared on an anion exchange cartridge (Sep-Pak QMA). Cells were radiolabelled and the labelling efficiency and efflux were evaluated. The in vivo biodistribution of copper-64-labelled WBC using [64Cu][Cu(oxinate)2] and [64Cu][Cu(tropolonate)2] was monitored in an infection and inflammation animal model using BALB/c mice. RESULTS: On-cartridge concentration of gallium-68, zirconium-89 and copper-64 enabled formation of oxine and tropolone tracers in small volumes with good yields (≥50%) and quality (extraction ≥90%). Prepared tracers radiolabelled the RBC comparable to indium-111 tracers and in vivo biodistribution of copper-64 labelled WBC showed clear accumulation of cells at the site of infection and inflammation. CONCLUSIONS: This on-cartridge preparation method enables simple formation of various PET tracers for cell radiolabelling. Zirconium-89 and copper-64 tracers radiolabelled cells with sufficient stability. Due to their longer half-life this approach could be promising for routine applications where longer evaluation periods for cell tracking are needed. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: This novel approach for on-cartridge concentration and preparation of oxine and tropolone precursors with different positron emitters, in small volume and suitable pH, offers a versatile tool towards cell labelling for preclinical and clinical PET applications.

Position paper on requirements for toxicological studies in the specific case of radiopharmaceuticals.

This is a position paper of the Radiopharmacy Committee of the EANM (European Association of Nuclear Medicine) addressing toxicology studies for application of new diagnostic and therapeutic radiopharmaceuticals (RP) that are not approved (i.e., not having a marketing authorization or a monograph in the European Pharmacopoeia), excluding endogenous and ubiquitous substances in humans. This paper discusses the requirements for clinical trials with radiopharmaceuticals for clinical research applications, not necessarily intended to aim at a marketing authorization. If marketing authorization is intended, scientific advice of the competent authorities is mandatory and cannot be replaced by this position paper. The position paper reflects the view of the Radiopharmacy Committee of the EANM and can be used as a basis for discussions with the responsible authorities.

Expression of somatostatin receptor subtype 2 and subtype 5 in thyroid malignancies.

AIM: To retrospectively analyse the expression of somatostatin receptor subtypes 2 (SSTR 2) and 5 (SSTR 5) in thyroid malignancies, possibly the most relevant subtypes for targeted therapy with somatostatin peptide radioligands. In addition, findings were also correlated with the course of disease. PATIENTS, METHODS: 87 consecutive patients (59 women, 28 men) with thyroid malignancy were included; 52 had papillary carcinoma, 24 follicular carcinoma, six medullary carcinoma, two poorly differentiated carcinoma and three anaplastic carcinoma. After initial therapy 70 (80.5%) patients showed complete remission, 11 (12.6%) patients partial remission with clinical and biochemical signs of residual disease and six (6.9%) patients progressive disease. The immunohistochemical staining results of the primary malignancy for SSTR 2 and SSTR 5 were semiquantitatively assessed and correlated with various outcome parameters. RESULTS: In 10 of 87 (11.49%) thyroid cancer samples SSTR 2 showed positive immunohistochemical expression as compared to 75 of 87 (86.20%) for SSTR 5. All SSTR 2-positive cases expressed SSTR 5. Persistent or recurrent disease was found in 17 of 87 cases (19.54%). Fifty percent (6 /12) of SSTR 5-negative patients showed persistent disease as compared to 14.7 % (11 / 75) of SSTR 5-positive patients: seven of these were exclusively SSTR 5-positive, 4 showed dual expression of SSTR 5 and SSTR 2 (p = 0.01). No case showed only SSTR 2 expression. CONCLUSIONS: SSTR 5 was shown to be the main receptor subtype in the analysed differentiated or anaplastic thyroid malignancies, whereas SSTR 2 was found only in a small percentage. Deficient SSTR expression may indicate higher risk for persistent or recurrent disease after initial therapy. For this reason immunohistochemistry can be considered a prognostic marker which should be further validated in prospective studies.

68Ga-DOTA-TOC uptake in neuroendocrine tumour and healthy tissue: differentiation of physiological uptake and pathological processes in PET/CT.

PURPOSE: We wanted to establish the range of (68)Ga-DOTA-TOC uptake in liver and bone metastases of patients with neuroendocrine tumours (NET) and to establish the range of its uptake in pancreatic NET. This would allow differentiation between physiological uptake and tumour-related somatostatin receptor expression in the pancreas (including the uncinate process), liver and bone. Finally, we wanted to test for differences in patients with NET, either treated or not treated with peptide receptor radionuclide therapy (PRRT). METHODS: In 249 patients, 390 (68)Ga-DOTA-TOC PET/CT studies were performed. The clinical indications for PET/CT were gastroenteropancreatic NET (194 studies), nongastroenteropancreatic NET (origin in the lung and rectum; 46 studies), NET of unknown primary (111 studies), phaeochromocytoma/glomus tumours (18 studies), and radioiodine-negative metastatic thyroid carcinoma (21 studies). RESULTS: SUVmax (mean ± standard deviation) values of (68)Ga-DOTA-TOC were 29.8 ± 16.5 in 162 liver metastases, 19.8 ± 18.8 in 89 bone metastases and 34.6 ± 17.1 in 43 pancreatic NET (33.6 ± 14.3 in 30 tumours of the uncinate process and 36.3 ± 21.5 in 13 tumours of the pancreatic tail). A significant difference in SUVmax (p < 0.02) was found in liver metastases of NET patients treated with PRRT. There were significant differences in SUVmax between nonmalignant and malignant tissue for both bone and liver metastases and for pancreatic NET including the uncinate process (p < 0.0001). At a cut-off value of 17.1 the specificity and sensitivity of SUVmax for differentiating tumours in the uncinate process were 93.6 % and 90.0 %, respectively (p < 0.0001). CONCLUSION: (68)Ga-DOTA-TOC is an excellent tracer for the imaging of tumours expressing somatostatin receptors on the tumour cell surface, facilitating the detection of even small tumour lesions. The noninvasive PET/CT approach by measurement of regional SUVmax can offer important clinical information to distinguish between physiological and pathological somatostatin receptor expression, especially in the uncinate process. PRRT does not significantly influence SUVmax, except in liver metastases of patients with NET.

Full automation of (68)Ga labelling of DOTA-peptides including cation exchange prepurification.

Here we describe a fully automated approach for the synthesis of (68)Ga-labelled DOTA-peptides based on pre-concentration and purification of the generator eluate by using a cation exchange-cartridge and its comparison with fully automated direct labelling applying fractionated elution. Pre-concentration of the eluate on a cation exchange cartridge both using a resin-based and a disposable cation-exchange cartridge efficiently removed (68)Ge as well as major metal contaminations with Fe and Zn. This resulted in a high labelling efficiency of DOTA-peptides at high specific activity (SA) with short synthesis times.

Comparison of in vitro and in vivo properties of [99mTc]cRGD peptides labeled using different novel Tc-cores.

AIM: The alfa(v)beta(3) integrin is involved in angiogenesis and tumor metastasis. Arginine-glycine-aspartic acid (RGD)-peptides bind with high affinity to this integrin. This study compares the influence of (99m)Tc-labeling applying novel Technetium-cores on imaging characteristics of the radiolabeled peptide. METHODS: Different peptide conjugates based on the cyclic pentapeptide c(RGDyK) (cRGD) were prepared and characterized (HYNIC-, Cys-, L2- and Pz1-cRGD). Radiolabeling experiments using different coligands for HYNIC-cRGD, the (99m)Tc(CO)(3) metal fragment for PZ-1-cRGD (pyrazolyl-derivative), the Tc-nitrido-core using a phosphine-coligand (PNP) for Cys-cRGD and an isonitrile-conjugate (L2-cRGD) together with a NS(3)-coligand (4+1 concept) were performed and showed labeling yields >90% at high specific activities. RESULTS: A high in vitro stability was observed, plasma protein binding and lipophilicity varied considerably between different radiolabeled cRGD conjugates. Experiments on biological activity of the radiolabeled peptides using alfa(v)beta(3) positive (M21) and negative (M21L) tumor cells did show specific uptake of various conjugates. Studies in tumor bearing animals revealed significant differences between different conjugates concerning pharmacokinetic behavior (predominant renal excretion to considerable hepatobiliary clearance) as well as tumor uptake (0.2-2.7%ID/g). Highest specific tumor uptake and tumor/background values were found for [(99m)Tc]EDDA/HYNIC-c(RGDyK), [(99m)Tc]Nitrido-PNP-Cys-c(RGDyK) and [(99m)Tc(CO)(3)]-Pz1-c(RGDyK). CONCLUSIONS: Using novel Tc-cores such as the (99m)Tc(CO)(3) metal fragment, Tc-nitrido- and the 4+1 concept peptides could be labeled with [(99m)Tc]technetium at high specific activities resulting in complexes with high stability, but binding moieties have to be optimized especially concerning hydrophilicity resulting in renal rather than hepatobiliary excretion. This comparative study underlines that peptide labeling strategies using (99m)Tc have to be properly selected and optimized. Different in vitro assays are necessary to predict targeting properties in vivo.

99mTc-labelled HYNIC-minigastrin with reduced kidney uptake for targeting of CCK-2 receptor-positive tumours.

PURPOSE: Different attempts have been made to develop a suitable radioligand for targeting CCK-2 receptors in vivo, for staging of medullary thyroid carcinoma (MTC) and other receptor-expressing tumours. After initial successful clinical studies with [DTPA(0),D: Glu(1)]minigastrin (DTPA-MG0) radiolabelled with (111)In and (90)Y, our group developed a (99m)Tc-labelled radioligand, based on HYNIC-MG0. A major drawback observed with these derivatives is their high uptake by the kidneys. In this study we describe the preclinical evaluation of the optimised shortened peptide analogue, [HYNIC(0),D: Glu(1),desGlu(2-6)]minigastrin (HYNIC-MG11). METHODS: (99m)Tc labelling of HYNIC-MG11 was performed using tricine and EDDA as coligands. Stability experiments were carried out by reversed phase HPLC analysis in PBS, PBS/cysteine and plasma as well as rat liver and kidney homogenates. Receptor binding and cell uptake experiments were performed using AR4-2J rat pancreatic tumour cells. Animal biodistribution was studied in AR4-2J tumour-bearing nude mice. RESULTS: Radiolabelling was performed at high specific activities and radiochemical purity was >90%. (99m)Tc-EDDA-HYNIC-MG11 showed high affinity for the CCK-2 receptor and cell internalisation comparable to that of (99m)Tc-EDDA-HYNIC-MG0. Despite high stability in solution, a low metabolic stability in rat tissue homogenates was found. In a nude mouse tumour model, very low unspecific retention in most organs, rapid renal excretion with reduced renal retention and high tumour uptake were observed. CONCLUSION: (99m)Tc-EDDA-HYNIC-MG11 shows advantages over (99m)Tc-EDDA-HYNIC-MG0 in terms of lower kidney retention with unchanged uptake in tumours and CCK-2 receptor-positive tissue. However, the lower metabolic stability and impurities formed in the labelling process still leave room for further improvement.

Evidence for Somatostatin receptor 2 in thyroid tissue.

Somatostatin receptor scintigraphy has found considerable interest for imaging thyroid tumours. Recently, also therapeutic application of Somatostatin analogues labelled with beta-emitting radionuclides has been suggested as treatment option for thyroid tumours with absent radioiodine uptake. Most of the radiolabelled analogues available show a predominant affinity for Somatostatin receptor subtype 2. This study reports on the in vitro characterisation of Somatostatin receptor subtype mRNAs in thyroid tumours and normal thyroid tissue by means of RT-PCR. Surgical samples of 21 patients were collected, and mRNA of 16 tumour and 17 control specimen was isolated. mRNA expression for Somatostatin, SSTR subtype 1-5, thyroid markers (NIS, TSH, Tg, TPO) and control markers (GAPDH, beta-actin) was determined. PCR results were correlated with immunohistochemistry staining using SSTR2 receptor specific antibodies. 94% of all samples expressed Somatostatin receptor mRNA with predominant expression of subtype 2, less predominant of subtype 5 and subtype 3. Somatostatin receptor subtype 2 mRNA expression correlated well with immunohistochemical staining pattern in 13/16 samples, SSTR2 immunohistochemistry was positive in 87% of the samples. Our results show that Somatostatin receptor 2 is predominantly expressed on thyroid tissue and is a valid target for treatment of thyroid tumours. Octreotide derivatives currently used in Nuclear medicine seem to be well suited to target receptors expressed in thyroid tumours.

99mTc-EDDA/HYNIC-Tyr(3)-octreotide for staging and follow-up of patients with neuroendocrine gastro-entero-pancreatic tumors.

AIM: To evaluate the use of 99mTc-EDDA-hydrazinonicotinyl-Tyr3-octreotide (Tc-TOC) for staging and follow-up of neuroendocrine gastro-entero-pancreatic (GEP) tumors with special focus on the acquisition protocol including single photon emission computed tomography (SPECT). METHODS: Eighty-eight patients (37 female, 51 male; age range: 16 to 81 years; mean age: 56.3 years) were studied: 42 patients for staging after initial histological confirmation and 46 patients during post-therapy follow-up. An average activity of 400 MBq of the radiopharmaceutical was injected. All tumors originated from neuroendocrine tissue of the gastroenteropancreatic tract. Whole body scintigrams at 4 h postinjection and SPECT of the abdomen were obtained in all patients. Additional planar images of the abdomen were acquired at 2 h after injection in 68 patients. RESULTS: The Tc-TOC scan result was true-positive in 56 patients, true-negative in 17, false-negative in 14, and false-positive in 1 patient. The false-positive finding was caused by a colonic adenoma. Overall, a scan sensitivity of 80% (56/70 patients), specificity of 94.4% (17/18 patients) and accuracy of 82.9% (73/88 patients) were calculated on patient basis. In total, Tc-TOC detected 357 foci in 69 patients. In 7 patients equivocal findings were observed in the bowel at 4 h postinjection without corresponding tracer uptake in the scan 2 h earlier, meaning that these abnormal findings were correctly classified as non-malignant. In addition to planar views, SPECT revealed further 62 lesions. CONCLUSIONS: Tc-TOC with one-day, dual-time acquisition protocol is an accurate staging procedure in patients with neuroendocrine GEP tumors. SPECT shows high sensitivity for detection of abdominal lesions, while earlier images improve the reliability of abnormal abdominal findings.

99mTc-labeling and in vitro and in vivo evaluation of HYNIC- and (Nalpha-His)acetic acid-modified [D-Glu1]-minigastrin.

Gastrin/CCK-2 receptors are overexpressed in a number of tumors such as medullary thyroid cancer (MTC) and small cell lung cancer (SCLC). Recently [D-Glu1]-minigastrin (MG) has been radiolabeled with 131I, 111In, and 90Y and evaluated in patients. This study describes the labeling and evaluation of MG with technetium-99m using two different labeling approaches: HYNIC as bifunctional coupling agent and (Nalpha-His)Ac as tridentate ligand for 99mTc(CO3) labeling. Labeling was perfomed at high specific activities using Tricine and EDDA as coligands for HYNIC-MG and [99mTc(OH2)3(CO)3]+ for (Nalpha-His)Ac-MG. Stability experiments were carried out by reversed phase HPLC analysis in PBS, serum, histidine, and cysteine solutions, as well as rat liver and kidney homogenates. Receptor binding and internalization experiments were performed using CCK-2 receptor positive AR42J rat pancreatic tumor cells. Biodistribution experiments were carried out in nude mice carrying AR42J tumors by injection of 99mTc-labeled peptide with or without coinjection of 50 microg of minigastrin I human (MGh). HYNIC-MG and (Nalpha-His)Ac-MG could be radiolabeled at high specific activities (>1 Ci/micromol). For HYNIC-MG, high labeling yields (>95%) were achieved using Tricine and EDDA as coligands. Stability experiments of all 99mTc-labeled conjugates revealed a high stability of the label in PBS and serum as well as toward challenge with histidine and cysteine. Incubation in kidney homogenates resulted in a rapid degradation of all conjugates with <10% intact peptide after 60 min at 37 degrees C, with no considerable differences between the radiolabeled conjugates; a somewhat lower degradation rate was seen in liver homogenates. Protein binding varied considerably with lowest levels for 99mTc-EDDA/HYNIC-MG. Competition experiments of unlabeled conjugates on AR42J membranes versus [125I-Tyr12]-gastrin I showed high CCK-2 receptor affinity for all conjugates under study. Internalization behavior was very rapid for all radiolabeled conjugates in the order of 99mTc-(Nalpha-His)Ac-MG > 99mTc-EDDA/HYNIC-MG > 99mTc-Tricine/HYNIC-MG. In tumor-bearing nude mice the highest tumor-uptake was observed with 99mTc-EDDA/HYNIC-MG (8.1%ID/g) followed by 99mTc-Tricine/HYNIC-MG (2.2%ID/g) and 99mTc-(Nalpha-His)Ac-MG (1.2%ID/g) which correlated with kidney uptake (101.0%ID/g, 53.8%ID/g, 1.8%ID/g respectively). In this series of compounds 99mTc-EDDA/HYNIC-MG with its very high tumor/organ ratios except for kidneys seems to be the most promising agent to target CCK-2 receptors. Despite promising properties concerning receptor binding, internalization, and in vitro stability, 99mTc-(Nalpha-His)Ac-MG showed low tumor uptake in vivo.

The influence of chelator on the pharmacokinetics of 99mTc-labelled peptides.

Because of the ideal properties of technetium-99m for imaging, more methods have been developed for labelling peptides and other biomolecules with this radionuclide that any other. While few detailed comparative studies have been performed, it has become apparent that the use of different labelling procedures can exert a profound effect on the pattern of biodistribution after intravenous administration of the radiotracer. The most significant influence of the labelling method is on the rate and route of excretion of the radionuclide. While some procedures tend to direct excretion towards the hepatobiliary route, others tend to favour renal clearance. Although the factors which exert this influence are still not fully understood, it is clear that the charge, lipophilicity and stability of the technetium-peptide complexes play major roles. A greater understanding of these factors will allow the development of improved radiotracers which demonstrate improved targeting potential as a result of lower uptake and consequent radiation dose by normal tissues.

Surfactant protein B labelled with [(99m)Tc(CO)3(H20)3](+) retains biological activity in vitro..

UNLABELLED: Labelling of the hydrophobic surfactant protein B (SP-B) under non-reducing conditions was achieved with [(99m)Tc(CO)(3)(H2O)(3)](+) prepared according to Alberto et al. (JACS, 1998). The binding of radioactivity was protein-specific, with an overall radiochemical yield of 50%. Gel electrophoresis and Westernblot analyses showed no structural changes of SP-B. Spreading properties and surface activity of (99m)Tc-labelled SP-B in an air/water interface coincided with those of unlabelled SP-B. (99m)Tc-SP-B seems to be a promising agent to observe surfactant spreading under clinical conditions. BACKGROUND: Therapeutic results for surfactant instillation in clinical trials are conflicting. The (99m)Tc-labelling of surfactant would allow to observe its spreading in the lung under clinical conditions. METHODS: [(99m)Tc(CO)(3)(H2O)(3)](+) was prepared as described by Alberto et al. (JACS, 1998). This carbonyl complex was used for the direct labelling of surfactant protein B (SP-B) under non-reductive conditions by direct incubation with SP-B at elevated temperature followed by extraction into CHCl(3)/MeOH. RESULTS: The hydrophobic protein SP-B was labelled with [(99m)Tc(CO)(3)(H2O)(3)](+). An overall radiochemical yield of about 50% was achieved. HPLC-analysis revealed a single radiolabelled species according to UV elution profile of SP-B, supported by paper and size exclusion chromatography. Gel electrophoresis confirmed that the dimer structure of SP-B was preserved. Spreading properties of (99m)Tc-labelled SP-B in an air/water interface coincided with those of unlabelled SP-B. Spreading of radioactivity observed in a glass trough of 26 cm x 27 cm with a gamma camera was completed during the first 7-9 sec after application of (99m)Tc-labelled SP-B. The corresponding decrease of surface tension to 45 mN/m at the peripheral surface tension sensors took 7 sec +/- 2 sec (MEAN +/- STD; n = 3). CONCLUSIONS: Direct and specific (99m)Tc-labelling of the hydrophobic surfactant protein B was achieved using the [(99m)Tc(CO)(3)(H2O)(3)](+) precursor. This procedure can easily be used to prepare specifically labelled surfactant mixtures with spreading properties that coincide with those of unlabelled surfactant.

99mTc-EDDA/HYNIC-TOC: a new 99mTc-labelled radiopharmaceutical for imaging somatostatin receptor-positive tumours; first clinical results and intra-patient comparison with 111In-labelled octreotide derivatives.

[111In-diethylene triamine penta-acetic acid-D-Phe1]-octreotide (DTPA-octreotide) scintigraphy has gained widespread acceptance as a diagnostic clinical procedure in oncology for imaging somatostatin receptor-positive tumours. However, indium-111 as a radiolabel has several drawbacks, including limited availability, suboptimal gamma energy and high radiation burden to the patient. We have recently reported on the preclinical development of 99mTc-EDDA/HYNIC-TOC, a new octreotide derivative which showed promising results both in vitro and in vivo. We now report our initial clinical experiences with this new radiopharmaceutical in ten oncological patients. The clinical diagnoses were: carcinoid syndrome (n=5), thyroid cancer (n=3), pancreatic cancer (n=1) and pituitary tumour (n=1). The biodistribution and kinetics of 99mTc-EDDA/HYNIC-TOC were compared with those of 111In-DTPA-octreotide in six cases, and with those of 111In-DOTA-TOC in five cases. With the new tracer tumours were imaged within 15 min after injection and showed the highest target/non-target ratios 4 h after injection. Tumour uptake persisted up to 20 h p.i. The rate of blood clearance was similar to that of 111In-DTPA-octreotide but faster than that of 111In-DOTA-TOC, while urinary excretion was lower compared with the 111In derivatives. Semi-quantitative region of interest analysis showed that 99mTc-EDDA/HYNIC-TOC produced higher tumour/organ (target/non-target) ratios than the 111In derivatives, especially in relation to heart and muscle. Significantly more lesions could be detected in 99mTc images. We conclude that 99mTcEDDA/HYNIC-TOC shows better imaging properties for the identification of somatostatin receptor-positive tumour sites than currently available 111In-labelled octreotide derivatives.

Radiochemical purity of routinely prepared 99Tcm radiopharmaceuticals: a retrospective study.

Radiochemical purity is an important quality parameter for radiopharmaceuticals. In this study, the radiochemical purity of 2090 samples out of 7000 routine preparations of 20 different 99Tcm radiopharmaceuticals was tested using standard methods over a period of more than 7 years. The mean radiochemical purity was 96.92% (standard deviation = 6.71%). Seventy-four preparations failed to meet radiochemical purity limits; that is, 3.54% of all preparations tested or 1.06% of all preparations in the observation period. The reasons for substandard preparations were mainly related to laboratory-specific conditions. The introduction of a dedicated quality control protocol allowed the elimination of many sources of labelling failures and could reduce the number of administered preparations with an insufficient radiochemical purity. We stress the need for quality control in the preparation of radiopharmaceuticals and provide original radiochemical purity values of routinely prepared 99Tcm radiopharmaceuticals.