PET and SPECT imaging of a radiolabeled minigastrin analogue conjugated with DOTA, NOTA, and NODAGA and labeled with (64)Cu, (68)Ga, and (111)In.

Cholecystokinin-2 (CCK-2) receptors, overexpressed in cancer types such as small cell lung cancers (SCLC) and medullary thyroid carcinomas (MTC), may serve as targets for peptide receptor radionuclide imaging. A variety of CCK and gastrin analogues has been developed, but a major drawback is metabolic instability or high kidney uptake. The minigastrin analogue PP-F11 has previously been shown to be a promising peptide for imaging of CCK-2 receptor positive tumors and was therefore further evaluated. The peptide was conjugated with one of the macrocyclic chelators DOTA, NOTA, or NODAGA. The peptide conjugates were then radiolabeled with either (68)Ga, (64)Cu, or (111)In. All (radio)labeled compounds were evaluated in vitro (IC50) and in vivo (biodistribution and PET/CT and SPECT/CT imaging). IC50 values were in the low nanomolar range for all compounds (0.79-1.51 nM). In the biodistribution studies, (68)Ga- and (111)In-labeled peptides showed higher tumor-to-background ratios than the (64)Cu-labeled compounds. All tested radiolabeled compounds clearly visualized the CCK2 receptor positive tumor in PET or SPECT imaging. The chelator did not seem to affect in vivo behavior of the peptide for (111)In- and (68)Ga-labeled peptides. In contrast, the biodistribution of the (64)Cu-labeled peptides showed high uptake in the liver and in other organs, most likely caused by high blood levels, probably due to dissociation of (64)Cu from the chelator and subsequent transchelation to proteins. Based on the present study, (68)Ga-DOTA-PP-F11 might be a promising radiopharmaceutical for PET/CT imaging of CCK2 receptor expressing tumors such as MTC and SCLC. Clinical studies are warranted to investigate the potential of this tracer.

Resistance to the tyrosine kinase inhibitor axitinib is associated with increased glucose metabolism in pancreatic adenocarcinoma.

Alterations in energy (glucose) metabolism are key events in the development and progression of cancer. In pancreatic adenocarcinoma (PDAC) cells, we investigated changes in glucose metabolism induced by resistance to the receptor tyrosine kinase inhibitor (RTKI) axitinib. Here, we show that human cell lines and mouse PDAC cell lines obtained from the spontaneous pancreatic cancer mouse model (Kras(G12D)Pdx1-cre) were sensitive to axitinib. The anti-proliferative effect was due to a G2/M block resulting in loss of 70-75% cell viability in the most sensitive PDAC cell line. However, a surviving sub-population showed a 2- to 3-fold increase in [C-14]deoxyglucose ([C-14]DG) uptake. This was sustained in axitinib-resistant cell lines, which were derived from parental PDAC. In addition to the axitinib-induced increase in [C-14]DG uptake, we observed a translocation of glucose transporter-1 (Glut-1) transporters from cytosolic pools to the cell surface membrane and a 2-fold increase in glycolysis rates measured by the extracellular acidification rate (ECAR). We demonstrated an axitinib-induced increase in phosphorylated Protein Kinase B (pAkt) and by blocking pAkt with a phosphatidylinositol-3 kinase (PI3K) inhibitor we reversed the Glut-1 translocation and restored sensitivity to axitinib treatment. Combination treatment with both axitinib and Akt inhibitor in parental pancreatic cell line resulted in a decrease in cell viability beyond that conferred by single therapy alone. Our study shows that PDAC resistance to axitinib results in increased glucose metabolism mediated by activated Akt. Combining axitinib and an Akt inhibitor may improve treatment in PDAC.

VEGF(121)b, a new member of the VEGF(xxx)b family of VEGF-A splice isoforms, inhibits neovascularisation and tumour growth in vivo.

BACKGROUND: The key mediator of new vessel formation in cancer and other diseases is VEGF-A. VEGF-A exists as alternatively spliced isoforms - the pro-angiogenic VEGF(xxx) family generated by exon 8 proximal splicing, and a sister family, termed VEGF(xxx)b, exemplified by VEGF(165)b, generated by distal splicing of exon 8. However, it is unknown whether this anti-angiogenic property of VEGF(165)b is a general property of the VEGF(xxx)b family of isoforms. METHODS: The mRNA and protein expression of VEGF(121)b was studied in human tissue. The effect of VEGF(121)b was analysed by saturation binding to VEGF receptors, endothelial migration, apoptosis, xenograft tumour growth, pre-retinal neovascularisation and imaging of biodistribution in tumour-bearing mice with radioactive VEGF(121)b. RESULTS: The existence of VEGF(121)b was confirmed in normal human tissues. VEGF(121)b binds both VEGF receptors with similar affinity as other VEGF isoforms, but inhibits endothelial cell migration and is cytoprotective to endothelial cells through VEGFR-2 activation. Administration of VEGF(121)b normalised retinal vasculature by reducing both angiogenesis and ischaemia. VEGF(121)b reduced the growth of xenografted human colon tumours in association with reduced microvascular density, and an intravenous bolus of VEGF(121)b is taken up into colon tumour xenografts. CONCLUSION: Here we identify a second member of the family, VEGF(121)b, with similar properties to those of VEGF(165)b, and underline the importance of the six amino acids of exon 8b in the anti-angiogenic activity of the VEGF(xxx)b isoforms.

Bifunctional bisphosphonate complexes for the diagnosis and therapy of bone metastases.

Easily synthesised and structurally well-defined novel imaging/therapeutic radiopharmaceutical agents for bone metastases are described.

99mTc-interleukin-2 scintigraphy in normal subjects and in patients with autoimmune thyroid diseases: a feasibility study.

PURPOSE: Radiolabelled interleukin-2 is a radiopharmaceutical used for the study of chronic inflammatory processes. (123)I-labelled interleukin-2 has successfully been used in a large number of patients affected by several immune-mediated diseases. (123)I, however, is expensive and not readily available. We have, therefore, developed a method for labelling interleukin-2 with (99m)Tc to high specific activity based on the use of an N(3)S bifunctional chelating agent. In this paper, we describe the results obtained with (99m)Tc-interleukin-2 in a series of eight normal subjects and of 12 patients with autoimmune thyroid diseases. METHODS: Biodistribution, pharmacokinetics, haematological and systemic toxicity, radiation absorbed dose and in vivo targeting were studied. RESULTS: Results showed rapid plasma clearance of (99m)Tc-interleukin-2 with retention mainly in the kidneys. Biodistribution and kinetics were similar to that observed for (123)I-interleukin-2. No acute systemic toxicity was found; a small decrease in peripheral blood lymphocytes was observed in the first hours only in patients, but it was mild and transient. (99m)Tc-interleukin-2 accumulated, to varying extents, in the thyroid of all patients affected by autoimmune thyroid diseases but not in the thyroid of normal subjects. The effective dose equivalent of a diagnostic activity of (99m)Tc-interleukin-2 (185 MBq) was 1.35 mSv. No correlation was observed between thyroid autoantibodies and uptake of (99m)Tc-interleukin-2. CONCLUSIONS: The use of (99m)Tc-interleukin-2 is safe and simple; the favourable dosimetry and biodistribution and the rapid clearance make it potentially useful for the study of chronic inflammatory diseases such as autoimmune thyroid disease.

Pancreatic scintigraphy with 99mTc-interleukin-2 at diagnosis of type 1 diabetes and after 1 year of nicotinamide therapy.

BACKGROUND: To evaluate the clinical utility of pancreatic scintigraphy with 99mTc-interleukin-2 to identify Type 1 diabetic patients with pancreatic inflammation at diagnosis. METHODS: 99mTc-interleukin-2 scintigraphy was performed on 42 newly diagnosed Type 1 diabetic patients, before and after 1 year of treatment with nicotinamide (25 or 50 mg/kg/day) in addition to intensive insulin therapy. Metabolic status was monitored every 3 months for 1 year. Sixteen normal subjects were studied as control. RESULTS: Significant pancreatic accumulation of 99mTc-interleukin-2 was found in 31% of the patients at the time of diagnosis. Patients positive or negative for pancreatic accumulation of interleukin-2 scintigraphy did not show any difference in metabolic or immunologic parameters at diagnosis. Positive patients, however, showed higher C-peptide values at 3 months and lower insulin requirement at 1 year, compared to negative patients (insulin requirement (IR): 0.33+/-0.11 vs 0.67+/-0.24 IU/kg/day, positive vs negative patients; p=0.0001); patients positive to IL2 scintigraphy treated with nicotinamide at 25 mg/kg were the only group showing a significant reduction in IR 1 year after diagnosis (IRt0: 0.53+/-0.30 vs IRt12: 0.28+/-0.07 IU/kg/day; p=0.013). After 1 year, all the positive patients showed a significant decrease in pancreatic uptake of 99mTc-interleukin-2 (P/B: 7.87+/-2.28 at diagnosis vs 5.00+/-1.23 after 1 year; p<0.0001 paired t-test). CONCLUSION: 99mTc-interleukin-2 scintigraphy at diagnosis of Type 1 diabetes may identify patients with pancreatic inflammation. In such patients, treated with nicotinamide at 25 mg/kg, insulin requirement and pancreatic inflammation after 1 year were significantly reduced suggesting that IL2 scintigraphy may be of potential use for assessing the autoimmune phenomena in endocrine pancreas.

SPECT/CT imaging of oncolytic adenovirus propagation in tumours in vivo using the Na/I symporter as a reporter gene.

Oncolytic adenoviruses have shown some promise in cancer gene therapy. However, their efficacy in clinical trials is often limited, and additional therapeutic interventions have been proposed to increase their efficacies. In this context, molecular imaging of viral spread in tumours could provide unique information to rationalize the timing of these combinations. Here, we use the human sodium iodide symporter (hNIS) as a reporter gene in wild-type and replication-selective adenoviruses. By design, hNIS cDNA is positioned in the E3 region in a wild-type adenovirus type 5 (AdIP1) and in an adenovirus in which a promoter from the human telomerase gene (RNA component) drives E1 expression (AdAM6). Viruses show functional hNIS expression and replication in vitro and kinetics of spread of the different viruses in tumour xenografts are visualized in vivo using a small animal nano-SPECT/CT camera. The time required to reach maximal spread is 48 h for AdIP1 and 72 h for AdAM6 suggesting that genetic engineering of adenoviruses can affect their kinetics of spread in tumours. Considering that this methodology is potentially clinically applicable, we conclude that hNIS-mediated imaging of viral spread in tumours may be an important tool for combined anticancer therapies involving replicating adenoviruses

(99m)Tc-rituximab radiolabelled by photo-activation: a new non-Hodgkin's lymphoma imaging agent.

PURPOSE: Rituximab was the first chimeric monoclonal antibody to be approved for treatment of indolent B-cell non-Hodgkin's lymphoma (NHL). It is directed against the CD20 antigen, which is expressed by 95% of B-cell NHLs. The aim of this study was to explore the possibility of radiolabelling rituximab with (99m)Tc for use as an imaging agent in NHL for early detection, staging, remission assessment, monitoring for metastatic spread and tumour recurrence, and assessment of CD20 expression prior to (radio)immunotherapy. METHODS: Rituximab was purified from Mabthera solution (Roche), photo-activated at 302 nm by UV irradiation and radiolabelled with (99m)Tc. The effectiveness of the labelling method was evaluated by determination of the number of free thiol groups per photoreduced antibody, radiochemical purity and in vitro stability of (99m)Tc-rituximab. RESULTS: On average, 4.4 free thiol groups per photoreduced antibody were determined. Radiolabelling yields greater than 95% were routinely observed after storage of the photo-activated antibody at -80 degrees C for 195 days. The direct binding assay showed preserved ability of (99m)Tc-rituximab to bind to CD20, with an average immunoreactive fraction of 93.3%. The internalisation rate was proven to be low, with only 5.3% of bound (99m)Tc-rituximab being internalised over 4 h at 37 degrees C. CONCLUSION: Our results demonstrate that (99m)Tc-rituximab of high radiochemical purity and with preserved binding affinity for the antigen can be prepared by photoreduction and that the method shows good reproducibility. (99m)Tc-rituximab will be further explored as an imaging agent applicable in NHL for the purposes mentioned above.

Tositumomab and iodine I 131 tositumomab for recurrent indolent and transformed B-cell non-Hodgkin's lymphoma.

PURPOSE: An open-label phase II study was conducted at two centers to establish the efficacy and safety of tositumomab and iodine I 131 tositumomab at first or second recurrence of indolent or transformed indolent B-cell lymphoma. PATIENTS AND METHODS: A single dosimetric dose was followed at 7 to 14 days by the patient-specific administered radioactivity required to deliver a total body dose of 0.75 Gy (reduced to 0.65 Gy for patients with platelets counts of 100 to 149 x 10(9)/L). Forty of 41 patients received both infusions. RESULTS: Thirty-one of 41 patients (76%) responded, with 20 patients (49%) achieving either a complete (CR) or unconfirmed complete remission [CR(u)] and 11 patients (27%) achieving a partial remission. Response rates were similar in both indolent (76%) and transformed disease (71%). The overall median duration of remission was 1.3 years. The median duration of remission has not yet been reached for those patients who achieved a CR or CR(u). Eleven patients continue in CR or CR(u) between 2.6+ and 5.2+ years after therapy. Therapy was well tolerated; hematologic toxicity was the principal adverse event. Grade 3 or 4 anemia, neutropenia, and thrombocytopenia were observed in 5%, 45%, and 32% of patients, respectively. Secondary myelodysplasia has occurred in one patient. Four patients developed human antimouse antibodies after therapy. Five of 38 assessable patients have developed an elevated thyroid-stimulating hormone; treatment with thyroxine has been initiated in one patient. CONCLUSION: High overall and CR rates were observed after a single dose of tositumomab and iodine I 131 tositumomab in this patient group. Toxicity was modest and easily managed.

Radiolabelling of glycosylated MFE-23::CPG2 fusion protein (MFECP1) with 99mTc for quantitation of tumour antibody-enzyme localisation in antibody-directed enzyme pro-drug therapy (ADEPT).

MFECP1 is a glycosylated recombinant fusion protein composed of MFE-23, a high-affinity anti-carcinoembryonic antigen (CEA) single chain Fv (scFv), fused to the enzyme carboxypeptidase G2 (CPG2), and has been constructed for use in antibody-directed enzyme pro-drug therapy (ADEPT). Radiolabelling of glycosylated MFECP1 with technetium-99m was developed for the purpose of determining tumour localisation of MFECP1 in a phase I ADEPT clinical study. The method used was 99mTc-carbonyl [99mTc(H2O)3(CO)3]+ (abbreviated to TcCO) mediated labelling of 99mTc to the hexahistidine (His) tag of MFECP1. MFECP1 fusion protein was labelled with TcCO under a variety of conditions, and this was shown to be a relatively simple and robust method. Tissue biodistribution was assessed in a CEA-expressing LS174T (human colon carcinoma) nude mouse xenograft model. Tissues were taken at 1, 4 and 6 h for assessment of distribution of radioactivity and for measurement of CPG2 enzyme levels. The amount of radioactivity retained by the tumour proved to be an accurate estimation of actual measured enzyme activity, indicating that this radiolabelling method does not appear to damage the antibody-antigen binding or the enzyme activity of MFECP1. However, correlation between CPG2 enzyme activity and measured radioactivity in liver, spleen and kidney was poor, indicating retention of radioactivity in non-tumour sites but loss of enzyme activity. The high retention of technetium radioisotope in normal tissues may limit the clinical applicability of this radiolabelling method for MFECP1; however, these results suggest that this technique does have applicability for measuring the biodistribution of His-tagged recombinant proteins.

Radiolabelling of peptides for diagnosis and therapy of non-oncological diseases.

Radiolabelled peptides have significant potential as radiopharmaceuticals for the diagnosis and therapy of receptor-expressing diseases. Methods have been developed for labelling peptides with a variety of radionuclides having a broad range of chemical and physical properties. These methods include both direct (where the radionuclide is bound directly to one or more atoms of the peptide structure) and indirect techniques in which bifunctional coupling agents are employed. Although most commonly applied to date in the field of oncology, a significant number of applications in non-oncological diseases have also been proposed and these can be expected to expand as the technology progresses. An overview is presented of some peptide-receptor systems in radiopharmaceutical development and the techniques which have been employed to radiolabel these peptides with isotopes of iodine, yttrium, indium, gallium, copper and technetium. While many of the examples employed are derived from cancer related indications, identical radiopharmaceutical chemistry can also be applied to peptides with applications in the fields of immunology, infection and other inflammatory conditions.

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.

Optimization of the small-scale synthesis of DOTA-Tyr3 -octreotide.

The clinical potential of 111In and 90Y labelled 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid conjugated Tyr3-octreotide (DOTA-TOC) have been reported in a number of publications, and Phase II clinical trials of 90Y-DOTA-TOC are currently in progress. However, to date, only a summary of the large-scale preparation of these radiopharmaceuticals has been published. This publication aims to describe our experience of the small-scale synthesis of DOTA-TOC in the hope that this will assist others in the preparation of this and other similar radioconjugates. DOTA in the form of the tri-t-butyl ester was coupled to the Lys5 (BOC) protected Tyr3-octreotide in N,N-dimethylformamide or N-methyl-2-pyrolidinone, in a three-step reaction involving conjugation, using O-(7-azabenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) and diisopropylethyamine (DIPEA) as coupling reagents, deprotection with trifluoroacetic acid and HPLC purification of the conjugates. The product was obtained in final yields of 60+/-5%. The purified product was characterized by mass spectroscopy, showing a molecular weight of 1421.55+/-0.08. In somatostatin receptor binding assays, the unlabelled DOTA-TOC showed an effective displacement of 99mTc labelled HYNIC-TOC (where HYNIC is hydrazinonicotinamide) (IC50=0.31+/-0.07 nm), confirming the retention of receptor-binding affinity. The conjugate could be efficiently labelled with 111In by addition of 111InCl3 and ammonium acetate buffer pH5 and heating (95 degrees C, 20 min).

Axillary node status in breast cancer patients prior to surgery by imaging with Tc-99m humanised anti-PEM monoclonal antibody, hHMFG1.

In early breast cancer axillary nodes are usually impalpable and over 50% of such patients may have an axillary clearance when no nodes are involved. This work identifies axillary node status by imaging with a Tc-99m radiolabelled anti-Polymorphic Epithelial Mucin, humanised monoclonal antibody (human milk fat globule 1), prior to surgery in 30 patients. Change detection analysis of image data with probability mapping is undertaken. A specificity of 93% and positive predictive value of 92% (both 100% if a second cancer in the axilla with negative nodes is considered) were found. A strategy for combining negative imaging with the sentinel node procedure is presented.

Hybridization and cell uptake studies with radiolabelled antisense oligonucleotides.

BACKGROUND: Radiolabelled antisense oligonucleotides have been proposed as radiopharmaceuticals for imaging changes in the level of gene expression in vivo. This paper describes a study of the uptake of radiolabelled oligonucleotides in cell lines expressing different levels of the target mRNA. METHODS: A 15-mer phosphorodiester deoxyoligonucleotide antisense to c-myc was labelled with 99mTc and 32P. Hybridization and stability studies were performed in vitro. Cell uptake studies were carried out in a c-myc expressing transformed rat embryonic fibroblast cell-line, TGR-1, and a knock-out cell line HO15.19 which does not express c-myc. RESULTS: The oligonucleotides were efficiently labelled with both radionuclides and retained their ability to hybridize with their complementary mRNA when extracted from cell lines. The radiolabelled oligonucleotides were stable for a few hours in human serum. No statistically significant difference was found between the uptake of radioactivity by the two cell lines. CONCLUSIONS: Although able to bind efficiently to their target in cell-free systems, radiolabelled oligonucleotides may be prevented from performing effectively as radiopharmaceutical vectors by the barriers imposed by cell membranes and/or intracellular metabolism.