Study of the labeling of two novel RGD-peptidic derivatives with the precursor [99mTc(H2O)3(CO)3]+ and evaluation for early angiogenesis detection in cancer.

In the initial stages of tumor formation, overexpression of integrins identifying the RGD sequence (Arg-Gly-Asp) is observed. The aim of the present study was the synthesis and labeling of two novel RGD derivatives, via the precursor [99mTc(H2O)3(CO)3]+, as well as the radiochemical and radiopharmacological evaluation of the labeled products. The labeling led to the formation of a single product in each case (>98%), with noteworthy in vitro stability, fast blood clearance and elimination by the hepatobiliary and the urinary systems.

Fast cancer uptake of 99mTc-labelled bombesin (99mTc BN1).

In human blood, breakdown of gastrin-releasing peptide and other bombesin-related peptides occurs in less than 15 min. This quick enzymatic cleavage might impair the diagnostic use of labelled bombesin (BN). 99mTc-labelled bombesin (99mTc BN1) was injected intravenously and dynamic uptake data were acquired for diagnosing 26 cancers of different origin: 15 breast, 3 prostate, 5 colo-rectal, 1 pancreas, 2 small cell lung cancers and 1 gastrinoma. Background subtracted tumour uptake data were plotted against time and fitted with known mathematical functions. Twenty-three out of 26 cancers showed rapid increase of radioactivity followed by a radioactivity plateau, with some oscillations around the average plateau value. The time to 80% of max activity (T80) was the reference parameter to measure and to compare the uptake speeds. The slowest T80 was 7 min in one T1b breast cancer, gastrinoma reached T80 in 5 min and node-positive prostate cancers in 2 min. N+ breast cancers showed T80 at 3.62 +/- 0.75 min, N- breast cancers at 5.5 +/- 0.88 min (p < 0.02). When all the tumours were considered, N+ tumours showed T80 at 2.68 +/- 1.03 min and N- cancers at 5.5 +/- 0.82 min. In all the cancer types, the uptake of 99mTc BN was faster than 10 min. This result shows the ability of 99mTc BN to image tumours. The faster uptake by N+ versus N- cancers probably depends on the higher blood flow in N+ cancers.

Tridentate ligands containing the SNS donor atom set as a novel backbone for the development of technetium brain-imaging agents.

In developing 99mTc complexes as potential brain-imaging agents, we investigated the coordination chemistry of ligands containing sulfur and nitrogen donor atoms with the general formula R-CH2CH2N(CH2CH2SH)2 (R = C2H5S, (C2H5)2N). These ligands act as tridentate SNS chelates to the TcO3+ core, leaving open one coordination site cis to the oxo group. In reactions with the highly reactive [99TcOCl4]- precursor, this vacancy was occupied by a chlorine atom. When the ligands reacted in the presence of 4-methoxythiophenol, using 99Tc(V)-gluconate as precursor, the vacancy was filled with 4-methoxythiophenol, which acted as coligand. Thus neutral mixed ligand complexes of the general formula [TcO((SCH2CH2)2NCH2CH2R)X], where X = Cl or 4-methoxythiophenol, were synthesized. The complexes were characterized by UV-vis, IR, 1H NMR, crystallographic, and elemental analyses. The crystal structures of 3a (R = C2H5S, X = Cl) and 4b (R = (C2H5)2N, X = 4-methoxythiophenol) demonstrated that the coordination geometry is trigonal bipyramidal with the N1 and Cl or S3 occupying the apical positions and the basal plane defined by the S1 and S2 of the tridentate ligand and the oxo group. The complexes 4a(99mTc) (R = C2H5S, X = 4-methoxythiophenol) and 4b(99mTc) were prepared using 99mTc-glucoheptonate as precursor and were purified by HPLC. Biodistribution in mice showed high initial brain uptake (3.68% and 3.56% dose/organ for 4a(99mTc) and 4b(99m-Tc), respectively). Complex 4b(99mTc) displayed significantly higher brain/blood values and prolonged retention in brain as well. The results suggest that structural modifications based on configurations 4a,b may provide novel 99mTc brain-imaging agents with improved biological characteristics.

Radioimmunoscintigraphy and radioimmunotherapy in cancer: principles and application.

Radioimmunoscintigraphy (RIS) and radioimmunotherapy (RIT) are recent approaches in the diagnosis and treatment of cancer. They take advantage of the antibody specificity of tumor surface antigens and of the emitted radiation from suitable radioisotopes, as a means of imaging (RIS) or therapy (RIT). Research into RIS and RIT radiolabelled agents remains an ongoing process. Principles governing the choice of radionuclides, labelling protocols, antibody suitability, and optimization of "tumor to normal tissue ratios" are the same for both RIS and RIT. The investigational stages of the labelled product, prior to clinical application, are also the same. These stages include radiochemical and radiobiological evaluation as well as determination of immunoreactivity. Furthermore, RIS may be considered as the first stage in development, before progressing on to RIT. Differences between RIS and RIT are associated with the application of each technique, that is, the type of radiation emitted by the isotope, dosage regimens, haematopoetic toxicity and the appearance of human antimurine antibody response (HAMA). RIS has found widespread clinical application, detecting a variety of tumors. However, its potential lies in patient management and in detecting metastases. On the other hand RIT is still in its infancy. It appears promising, and for the moment is used as a complementary technique to surgery and/or chemotherapy in clinical trials on cancer treatment. Finally, incorporation of these basic principles arising from past experiences, into the design of RIT trials improve responses.

Samarium-153 and technetium-99m-labeled monoclonal antibodies in angiogenesis for tumor visualization and inhibition.

BACKGROUND: Angiogenesis is the development of new blood vessels from pre-existing ones. Vascular endothelial growth factor (VEGF) is one of the most important angiogenic activators. Our studies are focused on the detection of VEGF by use of radiolabeled anti-endothelial monoclonal antibodies, which have the ability to localize in newly-formed vasculature of a cancerous origin. MATERIALS AND METHODS: The anti-endothelial monoclonal antibody VG76e was labeled with Samarium-153 and Technetium-99m. Biodistribution of the radiolabeled species was assessed in normal female Swiss mice, while tumor uptake was also evaluated. RESULTS: VG76e was labeled with 99mTc and 153Sm, resulting in a single product with a labeling yield of over 95%. Biodistribution studies showed non-specific uptake in any organ, with elimination via the hepatobiliary system. Finally, satisfactory tumor uptake was observed for both radiolabeled derivatives. CONCLUSION: Monoclonal antibodies raised against epithelial growth factors or their receptors, when labeled with appropriate radionuclides, may be a useful tool for early tumor detection and eventually for therapy.

Technetium labeled bombesin-like peptide: preliminary report on breast cancer uptake in patients.

Bombesin-like peptides are neurotransmitters and cancer growth factors. Several tumors, breast cancer among them, show one or more than one of the three known bombesin receptors. We have synthesized and labeled with technetium 99m a new pentadecapeptide, analogue to the leu13 amphibian bombesin (99mTc BN). Labeling yield was 83 +/- 4%. Prone Scintimammography was performed on five patients affected by breast cancers (T categorization: two T1b and three T1c), after injecting 0.7 mg, 185 to 296 MBq (5 to 8 mCi) of the peptide. Total body scan did not show free technetium biodistribution. No adverse reaction was observed. Prone Scintimammography with 99mTc Sestamibi (99mTc SM) was also performed few days later. 99mTc BN detected all 5 cancers, whereas 99mTc SM only four: all the T1c and one T1b cancer. Two of them showed axillary node invasion that was detected by both the radiotracers. A fibroadenoma present on contralateral breast to the one with cancer, was not detected neither by 99mTc SM nor by 99mTc BN. Tumor/breast normal tissue ratio (T/B) was constantly higher with 99mTc BN than with 99mTc SM. Maximal T/B was measured as 1.79 with 99mTc SM and 2.25 with 99mTc BN 5 min after fast i.v. administration. In conclusion our 99mTc BN is taken up by primary breast cancer showing higher T/B than 99mTc SM (p < 0.01). In our limited scale, 99mTc BN appears to be safe and, in our limited scale, even more accurate than 99mTc SM for detecting breast cancer.

Synthesis, chemical, radiochemical and radiobiological evaluation of a new 99mTc-labelled bombesin-like peptide.

A new pentadecapeptide bombesin analogue was prepared by Fmoc synthesis, purified by HPLC and identified by electron ionization mass spectrometry. The biological activity of the new peptide was tested on isolated human colonic muscle cells and compared to native bombesin. Labelling of the new biomolecule with Tc-99m yielded a single radioactive species which remained stable at room temperature for eight hours. In a binding assay, the radiolabelled peptide showed high affinity for oat-cell carcinoma (Kd = 9.8 nM) and colorectal adenocarcinoma (Kd = 27.2 nM). Biodistribution studies, performed in normal rodents, indicated uptake by organs that normally express bombesin receptors, such as liver, intestines and kidneys. Scintigraphic studies, performed in nude mice transplanted with small cell lung carcinoma and colon cancer cells, showed significant tumor uptake two hours p.i. The new synthetic pentadecapeptide appears to have promise for several malignancies, including oat-cell lung carcinoma, colorectal cancer and gastroenteropancreatic (GEP) tumors.

Radiochemical and radioimmunological data of 99Tcm-anti-CEA labelled by two diverse methods.

The aim of this study was to make a comparative evaluation of a direct and an indirect method for the labelling of anti-CEA with technetium-99m (99Tcm). With the direct method, disulphide bridges were cleaved by the use of 2-mercaptoethanol as reductant, whereas with the indirect method, the antibody was coupled to 2-iminothiolane. In both cases, a preformed intermediate chelate was used for 99Tcm exchange. The radiochemical and radiobiological behaviour of the 99Tcm-labelled species were studied. Furthermore, the influence of the labelling systems on the integrity of monoclonal antibodies, as well as the ability of 99Tcm-anti-CEA to tag onto human cancer cells, was investigated for the two labelling systems. Both methods showed a high labelling yield and resulted in immunoreactive and stable derivatives. However, detailed electrophoretical and radiochemical data, as well as the cysteine challenge trial, indicated relatively greater stability for the 2-mercaptoethanol reduction procedure.

Labeling of monoclonal antibodies with 153Sm for potential use in radioimmunotherapy.

The labeling of a monoclonal (anti-CEA) and a polyclonal (IgG) antibody with 153Sm has been investigated, using the bicyclic anhydride of DTPA (cDTPAa) as the chelating agent. The radiochemical study was performed using a combination of radioanalytical techniques (gel filtration, HPLC, ITLC-SG and SDS-PAGE). Optimization of factors affecting labeling (pH, Ab, Ab-DTPA concentration, etc.) leads to a labeling yield higher than 90%. Biodistribution studies in normal mice showed slow blood clearance and high uptake into the liver, kidney and lungs.

A 3D high-resolution gamma camera for radiopharmaceutical studies with small animals.

The results of studies conducted with a small field of view tomographic gamma camera based on a Position Sensitive Photomultiplier Tube are reported. The system has been used for the evaluation of radiopharmaceuticals in small animals. Phantom studies have shown a spatial resolution of 2mm in planar and 2-3mm in tomographic imaging. Imaging studies in mice have been carried out both in 2D and 3D. Conventional radiopharmaceuticals have been used and the results have been compared with images from a clinically used system.

Technetium-99m labelling of human immunoglobulin and preliminary clinical evaluation in tumour patients.

In the present investigation we have human immunoglobulin labelled with 99Tc(m), applying two different systems. The radiochemical characteristics of the labelled antibody were studied by conventional, radioanalytical methods. Further on, pharmacokinetics of this 99Tc(m)-labelled biomolecule were investigated, by i.v. administration in women, checked for tumours of the ovaries, uterus and cervix. Scintigraphic findings were compared to the results of other imaging techniques (CT, US, X rays), as well as to surgical findings. Our studies indicated that 99Tc(m)-human immunoglobuline can be applied successfully for the scintigraphic detection of several malignant and benign tumours of the female genital system. Tumour accumulation is probably due to the activation of particular cells, as macrophages and lymphocytes, responsible for inflammatory and immunological responses.

High resolution small animal single photon emission computed tomography: uptake of [99mTc]bombesin and [123I]ioflupane by rat brain.

AIM: The aims of this study were: 1) to perform brain single photon emission computed tomography (SPECT) in anesthetized rats with high resolution cameras (HRC) equipped with parallel hole collimation resolution of about 1 mm (HRC1) and 2 mm (HRC2); 2) to assess when and with which radio-tracer HRC1 SPECT shows advantages over HRC2. METHODS: We used two multicrystal HRCs with parallel square hole collimators, whose pure tungsten septa closely fit the crystals, in turn matched with a 4 inch2 position sensitive photomultiplier. HRC1 showed 1.1 mm and HCR2 2.1 mm resolution at collimator contact. HRCs performed 180 degrees semi-circular orbits around the head of rats: image reconstruction occurred with ordered subsets expectation maximization algorithms. Resolution of SPECT was measured with a Derenzo Phantom, resulting 1.4 mm for HRC1 and 2.3 mm for HRC2. Three rats were studied with [(99m)Tc]HMPAO, 3 rats with [(99m)Tc]bombesin (BN) and 48 h later with [(123)I]ioflupane (DaTSCAN). SPECT studies were reviewed by two experienced operators. RESULTS: Technetium-99m-HMPAO SPECT showed similar images with HRC1 and HRC2. The uptake of BN by amygdale, hippocampus and olfactory tract was detected by both cameras. DaTSCAN SPECT with HRC1 showed detailed image of the tail of the caudatus: this image was not obtained with HRC2. DaTSCAN and BN SPECT showed amygdale with both HRCs. However, only the central nucleus of amygdale takes up DaTSCAN, whereas central, lateral and basolateral amygdaloid nuclei express BN receptors. Only HRC1 SPECT showed amygdale larger with BN than with DaTSCAN. CONCLUSION: Spatial resolution of 1.4 mm is appropriate to detect selected subcerebral structures.

Indium-111-labelled cationic complexes of aminothiols: structure-activity correlation.

In the present work a series of NxS2 ligands were synthesized, investigated for the formation of cationic, indium chelates and evaluated comparatively in experimental animals. The compounds under study formed indium complexes which were stable in vitro and presented myocardial uptake. Animal studies showed that the chemical structure of the ligand plays an important role in biodistribution. A system of one six-membered and two five-membered rings, formed by the metal and the donor atoms of the ligand, may constitute a basic structure for the development of new indium tracers with myocardial affinity.

Comparative evaluation of 99mTc-labeled aminothiols as possible brain perfusion imaging agents.

Several new 99mTc aminodithiols were prepared and evaluated comparatively in experimental animals. The ligands were diamine, triamine or tetramine dithiols. Substituents were either attached on one of the nitrogens or introduced in between the two nitrogens of diamino dithiol (DADT) backbone. 99mTc-derivatives prepared by coupling DADT to secondary amines via ethylene group showed in mice high initial brain uptake and significant retention in brain tissue. These preparations were mixtures of more than one 99mTc-complex differing in brain uptake and clearance from the brain. The highest brain retention (brain to blood ratio 2.53, 15 min p.i.) was achieved with the 99mTc-complex prepared by coupling DADT with ethylene pyrrolidine. Lengthening the chain between the nitrogens of DADT moiety by introducing methyl or amino alkyl groups resulted in 99mTc-complexes with poor brain accumulation.

Immunotargeting of urothelial cell carcinoma with intravesically administered Tc-99m labeled HMFG1 monoclonal antibody.

Ten patients with transitional cell carcinoma (TCC) of the bladder received 3-6 mCi of HMFG1 monoclonal antibody (MAb) intravesically. The antibody was labeled with Tc-99m using the 2-Iminothiolane method. All patients underwent transurethral resection of the bladder tumor within 12-20 h following intravesical administration of 99m-Tc-HMFG1. The presence of the radiolabeled MAb in the circulation was studied by measuring the radioactivity in the serum for a period up to 20 h. Three of 10 patients underwent immunoscintigraphy (SPECT) 2-3 h postadministration and cancerous areas could be easily localized. Biopsies were taken from the tumor sites as well as from normal bladder mucosa. Absolute uptake of the administered MAb expressed as percent administered dose/kg of tissue could be evaluated only in eight patients. Multiple specimen taken from various tumor sites in every patient gave a wide range of uptake values ranging from 0 to 9.29% adm. dose/kg, whereas normal tissue uptake values ranged from 0 to 1.63, respectively.

Immunolocalization of transitional cell carcinoma of the bladder with intravesically administered technetium-99m labelled HMFG1 monoclonal antibody.

The aim of this study was the immunolocalization of transitional cell carcinoma of the bladder with a radiolabelled murine tumour-associated monoclonal antibody and the measurement of the absolute uptake of the antibody by the tumour. Fourteen patients with transitional cell carcinoma of the bladder received 3-6 mCi (111-222 MBq) of technetium-99m labelled HMFG1 monoclonal antibody intravesically and one patient, 2 mCi (74 MBq) of iodine-131 labelled 11.4.1, which is a non-tumour-specific monoclonal antibody. Four of the 15 patients were evaluated with single-photon emission tomography (SPET) 1 1/2 to 2 h post administration. All patients underwent transurethral resection of the bladder tumour within 12-20 h following intravesical administration of the radiolabelled antibody. The radioactivity of biopsy specimens from normal urothelium and tumour areas were counted in a gamma counter. The mean uptake of the radiolabelled antibodies from normal and tumour sites was expressed as a percentage of the administered dose per kilogram of tissue. Conventional histology and immunohistochemistry using HMFG1 monoclonal antibody were performed on paraffin sections of the biopsy specimens. Although our results are preliminary, it can be concluded that: (a) bladder tumours are well imaged by SPET when using 99mTc-HMFG1; (b) intravesically administered radiolabelled antibody remains on the bladder tissue and does not escape into the systemic circulation; (c) the wide range of tumour uptake values (0%-9.3% administered dose/kg) observed probably can be attributed to heterogeneity of the antigenic expression of the tumour; (d) values of 99mTc-HMFG1 monoclonal antibody uptake by the tumour do not justify future attempts at radioimmunotherapy.