Iodine-123 salmon calcitonin, an imaging agent for calcitonin receptors: synthesis, biodistribution, metabolism and dosimetry in humans.

Calcitonin is used to reduce high serum calcium levels in patients with malignancy, and as therapy for osteoporosis and Paget's disease. Receptors for the peptide have been identified in some human cancer cells including those of lung, breast, bone, prostate, and medullary carcinoma of the thyroid, suggesting that an imaging agent for the receptors might be useful in nuclear oncology. A modified chloramine-T method was used to label a pharmaceutical form of salmon calcitonin (SCT) with iodine-123. Labelling can be performed within 5 min including purification, resulting in >95% radiochemical purity and 70% yield. Digestion analysis shows labelling with two iodine atoms on the tyrosine residue. A Chinese hamster ovary cell-based assay showed that the receptor binding and activation were not impaired by the labelling. Biodistribution in mice was similar to that of commercially available mono-iodinated 125I-labelled SCT, kidney being the principal target organ. Evaluation in three patients previously diagnosed as having Paget's disease (injected with 37 MBq [123I]diiodotyrosyl22-SCT, containing less than 4 IU hormone, imaged dynamically up to 0.5 h and at intervals up to 24 h) shows early uptake in liver, kidney and sites of known Paget's disease but not in normal bone, and later uptake in thyroid and stomach. Blood clearance was fitted to a biexponential with half-lives of 3.4-7.4 min and 3-34 h. Radiation dosimetry was estimated using MIRDOSE 3. The highest doses (mean mGy/MBq) were to thyroid (6.8x10(-1)) and kidney (6.0x10(-2)), with a whole-body dose 3.0x10(-2). High performance liquid chromatography analysis revealed that urinary radioactivity was mostly in the form of iodide and diiodotyrosine within minutes of injection, indicating rapid in vivo breakdown. In summary, [123I]diiodotyrosyl22-SCT binds to calcitonin receptors and can image sites of Paget's disease but its imaging potential is not optimal because of rapid breakdown and clearance from target tissues, and an alternative radiolabelling approach is required.

In vitro and in vivo studies with pentavalent technetium-99m dimercaptosuccinic acid.

The purpose of this investigation was to characterise the in vivo chemistry and binding mechanisms of technetium-99m dimercaptosuccinic acid [99mTc(V)DMSA]. Biodistribution was studied in mice by frozen section whole-body autoradiography and microautoradiography in selected tissues. Binding to bone mineral analogues was studied in vitro using various forms of calcium phosphate and hydroxyapatite under varied conditions. Similar studies with 99mTc-hydroxymethylene diphosphonate (HDP) were also carried out for comparison. The in vivo stability of 99mTc(V)DMSA was monitored by high-performance liquid chromatographic analysis of blood and urine samples taken over 24 h from patients injected with the tracer. Whole-body autoradiography shows that 99mTc(V)DMSA has highest affinity for bone (cortical rather than medullary) in mice. Substantial uptake of the tracer was also observed in the kidney (cytoplasm of cortical renal tubular cells). No specific localisation was observed in the liver at either the microscopic or the macroscopic level. While 99mTc-HDP bound strongly to calcium phosphates under all conditions, 99mTc(V)DMSA binding was inhibited in the presence of phosphate and was stronger at pH 6.0 than at pH 7. 4. In non-phosphate buffers, however, the binding of 99mTc(V)DMSA remained high across the pH range 4-7.4. 99mTc(V)DMSA binds to calcium phosphates chemically unaltered, and no radioactive species other than the three isomers of 99mTc(V)DMSA were detected in blood or urine samples taken from patients up to 24 h after injection. 99mTc(V)DMSA is stable in vivo, and no conversion of the complex to other chemical species needs to be invoked to explain its uptake in bone metastases or soft tissue tumour. Bone affinity may be due to reversible binding of the unaltered complex to the mineral phase of bone.

Frozen section microautoradiography in the study of radionuclide targeting: application to indium-111-oxine-labeled leukocytes.

UNLABELLED: The microscopic biodistribution of radioactivity in tissues is important in determining microdosimetry. This study addresses the use of frozen section microautoradiography in studying the subcellular distribution of 111In in leukocytes labeled with 111In-oxine. METHODS: In conjunction with frozen section microautoradiography, computer image analysis methods were applied to the analysis and quantification of leukocyte sections and superimposed autoradiographs. Rapid cell fractionation was used to confirm the results. RESULTS: The emulsion (Ilford K2) response was linear over the concentration range investigated (0-33 MBq ml-1). Resolution of radionuclide distribution was better than 2 microns. The autoradiographs showed no dependence of radiolabel uptake on cell type. Classification of all cells into intervals according to grain density suggests an exponential rather than normal distribution, with approximately 50% of cells having little or no radiolabel. In any one sample, cells which were heavily labeled were approximately 10 times more likely to be found in aggregates (60% found in aggregates, mostly neutrophils) than cells which were not heavily labeled (6% found in aggregates); and the grain densities were at least twofold higher over nuclei than over cytoplasm. The last observation was confirmed by the rapid cell fractionation method which showed that approximately 57% of the total radioactivity was bound to nuclei. CONCLUSION: Frozen section microautoradiography is a practical and reliable approach to determining sub-cellular distribution of 111In. The radiolabeling process causes aggregation of neutrophils. Uptake is not significantly dependent on cell type, but only a fraction of cells are appreciably labeled. The radioactive concentration in cell nuclei is at least two-fold higher than in cytoplasm. Microautoradiography can be used to provide distribution data as input into computer models for sub-cellular dosimetry.

Labelling of leucocytes with colloidal technetium-99m-SnF2: an investigation of the labelling process by autoradiography.

Autoradiography of smears and frozen sections of labelled cell suspensions was used to study the distribution of radioactivity in and among blood cells labelled in either whole blood or leucocyte-rich plasma (LRP) with technetium-99m-SnF2 colloid. The tracer proved selective for neutrophils: the labelling probability (relative to that for erythrocytes) for each cell type in LRP (mean of five samples) was: neutrophils, 9.4; lymphocytes, 3.7; monocytes, 3.0; eosinophils 1.4; erythrocytes, 1.0. When labelling was carried out in whole blood (five samples), 74.5% +/- 8.3% of the cell-bound radioactivity was bound to erythrocytes, 13.6% +/- 6.5% to neutrophils, and 11.9% +/- 2.1% to lymphocytes, whereas in LRP (in which the leucocytes were only slightly outnumbered by erythrocytes), 76.5% +/- 14.9% of radioactivity was neutrophil bound. Labelled cells in smear autoradiographs exhibited two distinct silver grain patterns, "diffuse", consistent with an intracellular radioactive particle (in neutrophils), and "focal", consistent with a cell surface-adhering particle in direct contact with the emulsion (in other leucocyte types and erythrocytes). The phagocytic inhibitor cytochalasin B neither reduced the proportion of labelled neutrophils nor altered the labelling pattern. Neutrophils were able to scavenge radioactivity from the surface of erythrocytes. It is concluded that neutrophils bind 99mTc-SnF2 intracellularly by phagocytosis, with high affinity; other cells become labelled at the cell surface reversibly and with lower affinity. This selectivity is high enough to permit predominantly leucocyte labelling in LRP but not in whole blood.

Radionuclide targeting and dosimetry at the microscopic level: the role of microautoradiography.

The understanding of localisation mechanisms and microdosimetry of diagnostic and therapeutic radiopharmaceuticals depends on knowledge of their biodistribution at the microscopic level (cellular and subcellular) in the target tissues. Various methods have been advanced for obtaining information about this microdistribution: subcellular fractionation, secondary ion mass spectrometry imaging, microprobe elemental analysis in the electron microscope, and microautoradiography. This review compares these approaches, and discusses in detail the methodology of microautoradiography (the most generally useful approach) with imaging and therapy radionuclides. Literature examples of applications of microautoradiography in nuclear medicine are reviewed, and the future potential contribution of the techniques is assessed.

Autoradiography and density gradient separation of technetium-99m-exametazime (HMPAO) labelled leucocytes reveals selectivity for eosinophils.

Technetium-99m-Exametazime (HMPAO) is widely used for radiolabelling leucocytes for localization of infection. The subcellular distribution of radionuclide in the labelled cells and the distribution of radioactivity among the leukocyte population are incompletely understood. Frozen section autoradiography was used to determine quantitatively the distribution of 99mTc in leucocytes labelled with 99mTc-Exametazime. Sections of rapidly frozen suspensions of labelled leucocytes in plasma were autoradiographed on Ilford K2 emulsion and stained with haematoxylin and eosin. Neutrophils, eosinophils and mononuclear cells were separated by Percoll density gradient centrifugation. Cell nuclei were isolated by a rapid cell-breakage and fractionation method. In a typical experiment mean grain densities [grains/100 microns2 (ESD)] over cells were: eosinophils 31.2 (18.4), neutrophils 3.5 (3.5), mononuclear cells 4.2 (5.1). Mean grain numbers per cell (ESD) were: eosinophils 13 (6.8), neutrophils 1.3 (1.3), mononuclear cells 1.1 (1.3). These findings were confirmed by separation of labelled leucocytes on discontinuous density gradients. In four separation experiments, the mean activity-per-cell ratio for eosinophils to neutrophils was 10.1 (4.8):1, and for eosinophils to mononuclear cells, 14.1 (6.7):1. The subcellular distribution of the label was investigated using image analysis of autoradiographs and cell fractionation. This revealed no selectivity for nuclear or extranuclear compartments. It may be concluded that 99mTc-Exametazime has strong selectivity for eosinophils over other leukocytes but no selectivity for nuclear/cytoplasmic compartments.