Albumin-based nanoparticles as magnetic resonance contrast agents: I. Concept, first syntheses and characterisation.

To develop a platform for molecular magnetic resonance imaging, we prepared gadolinium-bearing albumin-polylactic acid nanoparticles in the size range 20-40 nm diameter. Iterative cycles of design and testing upscaled the synthesis procedures to gram amounts for physicochemical characterisation and for pharmacokinetic testing. Morphological analyses showed that the nanoparticles were spheroidal with rough surfaces. Particle sizes were measured by direct transmission electron microscopical measurements from negatively contrasted preparations, and by use of photon correlation spectroscopy; the two methods each documented nanoparticle sizes less than 100 nm and generally 10-40 nm diameter, though with significant intrabatch and interbatch variability. The particles' charge sufficed to hold them in suspension. HSA retained its tertiary structure in the particles. The nanoparticles were stable against turbulent flow conditions and against heat, though not against detergents. MRI imaging of liquid columns was possible at nanoparticle concentrations below 10 mg/ml. The particles were non-cytotoxic, non-thrombogenic and non-immunogenic in a range of assay systems developed for toxicity testing of nanoparticles. They were micellar prior to lyophilisation, but loosely structured aggregated masses after lyophilisation and subsequent resuspension. These nanoparticles provide a platform for further development, based on non-toxic materials of low immunogenicity already in clinical use, not expensive, and synthesized using methods which can be upscaled for industrial production.

Preliminary experience with (68)Ga-DOTA-lanreotide positron emission tomography.

AIM: Positron emission tomography (PET) of (68)Ga-radiolabelled (SST) somatostatin receptor (R) binding peptides has recently been evaluated in SSTR positive tumor patients. First promising results in lung and thyroid tumor patients with (111)In-DOTA-Lanreotide (DOTA-LAN) scintigraphy have been described. We report our first experience with (68)Ga-labeled DOTA-LAN. METHODS: Eleven patients (3 non small cell lung cancer [NSCLC], 3 small cell lung cancer [SCLC], 3 radioiodine negative thyroid cancer, 2 medullary thyroid cancer [MTC]) were investigated. After intravenous injection of 75-150 MBq (68)Ga-DOTA-LAN dynamic studies were acquired over the tumor site for the first 40 min with a dedicated PET scanner in 3 patients, and 2 partial body scans were acquired at 20 and 50 min p.i. in 2 patients. Whole body acquisitions at 90 min after injection were acquired in all 11 patients. Image reconstruction was performed by iterative reconstruction utilizing additional transmission scans for attenuation correction. Vital parameters were recorded during the PET study and up to 24 h p.i. Blood and urinary sampling was done up to 4 hr after tracer injection in 8 patients. PET results were compared to conventional imaging techniques (CIT), i.e. computed tomography (CT) and/or magnetic resonance imaging (MRI). In 5 patients, (68)Ga-DOTA-LAN was compared with 2-[(18)F]fluoro-2-deoxy-D-glucose ((18)F-FDG). RESULTS: After intravenous (i.v.) injection of (68)Ga-DOTA-LAN the radioactivity in the blood rapidly decreased to less then 20% of the injected dose (ID) within the first 20 min and further decreased to less than 9% ID after 4 h. A cumulative urinary excretion of (68)Ga-DOTA-LAN up to 29.2 + or - 13.2% ID at 4 h was found. No acute side effects were observed. Tumor sites were visualized already during the first min after injection. Comparison of positron emission tomography (PET) and CIT showed concordant results in 3/8 patients and partial concordant results in 5/8 patients with matched results for the primary/recurrent tumor, mediastinal lymph nodes, or adrenal gland metastases. Partial concordant results were seen for the lung, bone, liver and cervical lymph node metastases. Micronodular metastases of the lung and the cerebrum were not visualized by (68)Ga-DOTA-LAN PET. The maximal standardized uptake values of the lung and bone tumor lesions ranged from 6 to 8 g/ml at 90 min p.i.. CONCLUSIONS: (68)Ga-DOTA-LAN visualized the majority of tumor lesions. Further studies are required to assess the clinical value, and to obtain the best imaging protocol of this new PET SSTR tracer.

Individualized peptide-related-radionuclide-therapy concept using different radiolabelled somatostatin analogs in advanced cancer patients.

AIM: Over the last decade, somatostatin (SST) receptor (R)-positive tumors have been treated using either (90)Y-DOTA-TOC, (177)Lu-DOTA-TATE or (90)Y-DOTA-LAN/(177)Lu-DOTA-LAN, at the Innsbruck Medical University. This report presents data from the evaluation of the initial 100 patients receiving receptor mediated radionuclide therapy (PRRT) according to our protocol. METHODS: One-hundred patients with SSTR-positive tumors were treated (36 female, 64 male; mean age, 58 years; range, 13 to 84 years), including 68 patients with neuroendocrine tumors (NET), and patients with other non-neuroendocrine tumors, e.g. patients with radioiodine-negative thyroid carcinoma, refractory to conventional treatment modalities. Patients were selected based on high SSTR expression as assessed by (68)Ga-DOTA-TOC as first choice tracer for patients with NET, or (68)Ga-DOTA-LAN for patients with other tumor entities, or if the (68)Ga-DOTA-TOC PET was negative. Following positron emission tomography (PET), individual dosimetry was regularly performed using (111)In-labeled compounds. Therapy cycles were repeated every 10 weeks using either (90)Y-DOTA-TOC (3.7 GBq, 3-5 cycles) or (177)Lu-DOTA-TATE (7.4 GBq, 3-4 cycles). Thirteen patients received both and 5 patients even 3 different therapeutic compounds. Each patient received an amino acid solution (arginin, lysine) to reduce the kidney dose. Between the radioactive cycles a long-acting SST analog was applied. Dosages were individually adapted depending on several disease related factors. RESULTS: Overall, following PRRT partial remission (PR) was observed in 23 patients (23 %), minor remission (MR) in 10 (10 %), stable disease (SD) in 42 patients (42 %). Although 25 patients (25 %) showed progressive disease (PD), palliative care was provided in most of these patients. In the group treated with 90Y-DOTA-TOC, 12 patients showed PR, 3 MR, 32 SD and 13 had PD. In the group of patients treated with (177)Lu-DOTA-TATE, PR was observed in 15 patients, MR in 8, SD in 19 and PD in 13 patients. Severe side effects (WHO Grad 3 and 4) were seen in only 6 % of patients. Severe long-term nephrotoxicity was observed in none of the patients. These adverse reactions were especially seen in patients who were treated with high doses per cycle, in patients pre-treated with chemotherapy and in patients with low clinical performance. CONCLUSIONS: PRRT with differently labelled tracers (Y-90 or Lu-177) and different SST-analogs is generally well tolerated without serious side effects. These results favour the combined use of radiolabeled SST analogs providing a customized tumour targeting for size reduction and improvement of quality-of-life. Extended time intervals and reduced individual doses make sense in patients with advanced tumor stages, in case of moderate SSTR-expression, and in patients with higher age.

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.

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-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.