Evaluation of a technetium-99m labeled bombesin homodimer for GRPR imaging in prostate cancer

Multimerization of peptides can improve the binding characteristics of the tracer by increasing local ligand concentration and decreasing dissociation kinetics. In this study, a new bombesin homodimer was developed based on an ε-aminocaproic acid-bombesin(7–14) (Aca-bombesin(7–14)) fragment, which has been studied for targeting the gastrin-releasing peptide receptor (GRPR) in prostate cancer. The bombesin homodimer was conjugated to 6-hydrazinopyridine-3-carboxylic acid (HYNIC) and labeled with ^99mTc for SPECT imaging. The in vitro binding affinity to GRPR, cell uptake, internalization and efflux kinetics of the radiolabeled bombesin dimer were investigated in the GRPR-expressing human prostate cancer cell line PC-3. Biodistribution and the GRPR-targeting potential were evaluated in PC-3 tumor-bearing athymic nude mice. When compared with the bombesin monomer, the binding affinity of the bombesin dimer is about ten times lower. However, the ^99mTc labeled bombesin dimer showed a three times higher cellular uptake at 4 h after incubation, but similar internalization and efflux characters in vitro. Tumor uptake and in vivo pharmacokinetics in PC-3 tumor-bearing mice were comparable. The tumor was visible on the dynamic images in the first hour and could be clearly distinguished from non-targeted tissues on the static images after 4 h. The GRPR-targeting ability of the ^99mTc labeled bombesin dimer was proven in vitro and in vivo. This bombesin homodimer provides a good starting point for further studies on enhancing the tumor targeting activity of bombesin multimers.     

A pre-targeting strategy for imaging glucose metabolism using technetium-99m labelled dibenzocyclooctyne derivative

During the last four decades, nuclear medicine has undergone enormous growth, and positron emission tomography (PET) has been in the driving seat for most of the time. ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG) is the most widely used agent for the detection of hibernating myocardium and metabolically active cancer tissue. But its cost and limited availability are the main limitations. For a long time different researchers and groups of pharmacists have tried to label glucose with a cheaper and long-acting radionuclide like ^99mTc. However, they failed to achieve this goal owing to the chemical complexity of ^99mTc and the lack of maintaining the physiological activity of diagnostic compounds. A pre-targeting strategy based on strain-promoted [3 + 2] azide-alkyne cycloaddition (SPAAC) reaction was applied to solve this problem. Functional click synthons were synthesized: 2-azido-2-deoxy-d-glucose (GlucN₃) as a glucose analogue, and N- (2- (2- (2- (bis (pyridin-2-ylmethyl) amino) ethoxy) ethoxy) ethyl-2- (6H-11,12-didehydrodibenzo [a,e] cycloocten-5-ylideneaminooxy) acetamide (C7) as a ^99mTc(CO)₃ labeling and azido-binding group. The results of biodistribution experiments in mice bearing S180 tumor show the relatively high tumor/blood ratio (up to 2.95) and tumor/muscle ratio (up to 6.37), and both of them decreases significantly in the glucose blocking experiment. It indicates that GlucN₃ behaves similarly to glucose and that in vivo SPAAC reactions can occur effectively. It is supposed that this pre-targeting strategy can indeed enhance target specificity and may be used for glucose metabolism imaging in tumor diagnosis.     

A kit for labelling erythrocytes or leukocytes with technetium-99m

A pretinning method for labelling erythrocytes with technetium-99m (^99mTc) in vitro has been developed using a kit which contains stannous chloride stabilized with gentisic acid. Labelling efficiency was 97.3% (SD 1.4%) for 80 patients. The method requires less time than the Brookhaven kit and results in a smaller volume for reinjection but provides equivalent clinical results. We have previously shown that leukocytes labelled with ^99mTc using the same gentisic acid kit are clinically equivalent to those labelled with HMPAO; thus, the kit is versatile and cost-effective.     

Evaluation of reduction-mediated labelling of antibodies with technetium-99m

Monoclonal antibodies can be labelled with technetium-99m by prereduction of the antibody with 2-mercaptoethanol, then reduction of pertechnetate with an aliquot of a stannous kit, resulting in > 97% labelling without the need for further purification. The present work shows that equally high labelling can be obtained with a variety of weak ligands and that the optimum quantity of stannous chloride is 2–4 μg. Although the label was stable to challenge with excess DTPA, cysteine was able to remove a portion of the label. We have also shown that this technique works with the IgG_2a isotype in addition to the previously reported IgG₁ isotype. This approach is simple, convenient and reproducible, and warrants further clinical evaluation.     

Single isomer technetium-99m tamoxifen conjugates

To produce an imaging agent for breast cancer using a technetium-99m-labeled agent specific for estrogen receptors, an N(2)S(2) bifunctional chelator was conjugated to Z- and E-aminotamoxifens through an amide linker. These bioconjugates have been chelated with both technetium-99m and rhenium. For the Z-isomer, chelation with rhenium in the presence of sodium acetate yields a mixture of two isomers, anti and syn, in a 1:1 ratio and in the presence of hydroxide results in only the anti isomer. Both the Z- and E-tamoxifen conjugates have been chelated with technetium-99m at the tracer level yielding a single isomer product, which is assigned as anti based on chromatographic comparison to the rhenium complexes. Radiochemical yields were consistently greater than 80%, with Sep-Pak column purification yielding a final product with >99% radiochemical purity and no residual starting material. Both in vitro and in vivo biological evaluation of the tamoxifen chelates indicated very limited estrogen receptor binding.     

The use of diaminodithiol for labeling small molecules with technetium-99m

To investigate the labeling of small molecules with ^99mTc by the bifunctional chelate approach, we have synthesized both a fatty acid and an estrone derivative containing a chelator of the N₂S₂ type. In the case of the fatty acid, this was a diaminodithiol (DADT) while for the estrone, a diaminodisulfide (DADS) was attached. The estrone derivative (5-(2-methylene estrone 3-methyl ether)-3,3,10,10-tetramethyl-1, 2-dithia-5,8-diazacyclodecane hydrochloride, DADS-E) was prepared by alkylation of DADS while the fatty acid derivative (N-(11-undecanoic acid)-N,N′-bis(2-methyl-2-mercaptopropyl) ethylenediamine hydrochloride, DADT-FA) was synthesized by alkylation of DADS followed by reduction. DADS-E was labeled in ethanol at elevated temperatures while DADT-FA was labeled at room temperature, both by stannous reduction. Paper chromatography showed both to be labeled and reverse-phase HPLC showed multiple peaks for both. Serum stability studies were performed by incubation at 37 °C with aliquots removed at 1 min and 1 day for analysis by size-exclusion HPLC. Initially, little pertechnetate or binding to serum proteins was observed whereas after 1 day the majority of activity in both cases was protein bound with 20 and 38% pertechnetate appearing for DADT-FA and DADS-E respectively. In conclusion, small biologically active molecules may be labeled with ^99mTc through an attached diaminodithiol or diaminodisulfide group.     

An APD-based iterative reconstruction method for simultaneous technetium-99m/iodine-123 SPECT imaging

Dual-isotope SPECT (DI-SPECT) studies offer significant advantages over sequential scans, foremost among them faster acquisition and perfect image registration. However, reconstructed images may be affected by substantial cross-talk contamination rendering them inadequate for diagnosis. This effect is especially strong for isotopes with close photopeak energies, such as ^99mTc (140 keV) and ¹²³I (159 keV). In this paper we present an iterative DI-SPECT reconstruction method which includes accurate, analytically computed scatter corrections provided by the APD (analytical photon distribution) algorithm. This algorithm calculates first and second order Compton scatter (based on the Klein–Nishina formula) and first order Rayleigh scatter. Both self-scatter and cross-talk between the two isotopes are evaluated using patient specific attenuation maps and an initial activity distribution estimate. To validate our method we performed experiments using the Data Spectrum, Inc. thorax phantom and a SPECT/CT camera system. Reconstructed images demonstrate significant improvement in data quantitation. Their quantitative accuracy increases up to a factor of two, even for activity ratios which strongly enhance cross-talk effects and seriously degrade projections.     

Synthesis of a technetium-99m labeled tricyclic ganciclovir analog for non-invasive reporter gene expression imaging

A potential radiopharmaceutical and HSV1-TK substrate, 3-((1,3-dihydroxypropan-2-yloxy)-methyl)-6-(4-(3-((2-mercaptoethyl)(2-(2-mercaptoethyl-amino)-ethyl)amino)propoxy)phenyl)-3H-imidazopurin-9(5H)-one-oxo-technetium(V), was synthesized via a converging approach and its chemical structure was comparatively characterized with a non-radioactive analog. The final radiochemical purity and yield were 97 and 73%, respectively.     

Bombesin derivative radiolabeled with technetium-99m as agent for tumor identification

A bombesin derivative was successfully radiolabeled in high labeling yield. Biodistribution studies and scintigraphic images in Ehrlich tumor-bearing mice were performed. This compound showed high accumulation in tumor tissue with high tumor-to-muscle and tumor-to-blood ratios. Thus, ^99mTc-HYNIC-Bombesin_(7–14) could be used as an agent for tumor diagnosis.     

Synthesis and biodistribution studies of carbohydrate derivatives radiolabeled with technetium-99m

Three carbohydrate derivatives, MAG₃-Gl, MAG₃-Ga, MAG₃-NG, were synthesized and radiolabeled in high yields. These substances were injected in health Swiss mice and their biodistribution were evaluated. Among them, ^99mTc-MAG₃-Ga displayed higher accumulation in hepatic tissue, due to the presence of specific receptors in the liver for this carbohydrate. Thus, the use of ^99mTc-MAG₃-Ga to assess hepatic function can be considered.     

Novel Tat-peptide chelates for direct transduction of technetium-99m and rhenium into human cells for imaging and radiotherapy

Rapid and efficient delivery of radioactive metal complexes to the cell interior would enable novel applications in medical imaging and radiotherapy. Membrane permeant peptide conjugates incorporating HIV-1 Tat transactivation protein sequences (GRKKRRQRRR) and an appropriate peptide-based motif (epsilon-KGC) that provides an N(3)S donor core for chelating technetium and rhenium were synthesized. Oxotechnetium(V) and oxorhenium(V) Tat-peptide complexes were prepared by facile transchelation reactions with permetalates, tin(II) chloride and sodium glucoheptonate. RP-HPLC showed two major [(99m)Tc]Tat-peptide species (4) that differed in retention time by approximately 2 min corresponding to two [Re]Tat-peptide species (7) shown to have identical mass, consistent with formation of two isomers, likely the oxo-metal diastereomers. [(99m)Tc]Tat-peptides were stable to transchelation in vitro. In human Jurkat cells, [(99m)Tc]Tat-peptide 4 showed concentrative cell accumulation (30-fold greater than extracellular concentration) and rapid uptake kinetics (t(1/2) < 2 min) in a diastereomeric-comparable manner. Paradoxically, uptake was enhanced in 4 degrees C buffer compared to 37 degrees C, while depolarization of membrane potential as well as inhibition of microtubule function and vesicular trafficking showed no inhibitory effect. Cells preloaded with 4 showed rapid washout kinetics into peptide-free solution. Modification of [(99m)Tc]Tat-peptide by deletion of the N-terminus Gly with or without biotinylation minimally impacted net cell uptake. In addition, the C-terminus thiol of the prototypic Tat-peptide was labeled with fluorescein-5-maleimide to yield conjugate 8. Fluorescence microscopy directly localized conjugate 8 to the cytosol and nuclei (possibly nucleolus) of human Jurkat, KB 3-1 and KB 8-5 tumor cells. Preliminary imaging studies in mice following intravenous administration of prototypic [(99m)Tc]Tat-peptide 4 showed an initial whole body distribution and rapid clearance by both renal and hepatobiliary excretion. Analysis of murine blood in vivo and human serum ex vivo revealed >95% intact complex, while murine urine in vivo showed 65% parent complex. Thus, these novel Tat-peptide chelate conjugates, capable of forming stable [Tc/Re(V)]complexes, rapidly translocate across cell membranes into intracellular compartments and can be readily derivatized for further targeted applications in molecular imaging and radiotherapy.     

Preparation and evaluation of technetium-99m labeled cardiac glycoside derivatives as potential myocardial imaging agent

Three cardiac glycosides, two natural, cymarin and convallotoxin and one synthetic, strophanthidin-β-d-glucoside were converted to their thiosemicarbazone and subsequently radiolabeled with ^99mTc by chelation. The resulting radioactive chelate complexes were evaluated in animals to determine the suitability of this class of compounds for myocardial imaging. It was observed from the animal biodistribution data of the three radioactive compounds, there was a considerable variation in the heart to non-target organ uptake ratio. A possible explanation of this variation was offered in the light of their lipophilic character, protein binding ability and affinity towards non-target receptors. It is anticipated that this study may help to develop a ^99mTc-cardiac glycoside complex with better distribution characteristics, and such a compound may offer a suitable alternative to ²⁰¹Tl, which is at present used for myocardial imaging.     

Radiolabeling of folic acid-modified chitosan with (99m)Tc as potential agents for folate-receptor-mediated targeting

The feasibility of chitosan (CS) as a backbone for the design of (99m)Tc-labeled targeting agent was evaluated in this study. Chitosan-folate conjugate (CSFA) and chitosan-folate dithiocarbamate (CSFADTC) were synthesized, characterized and radiolabeled with (99m)Tc as model compounds for folate-receptor (FR) targeting. (99m)Tc-complexes were prepared with high radiochemical purity and high stability. The hydrophilicities of these (99m)Tc-complexes were determined by partition coefficient experiments. The results of biodistribution in normal mice showed that the folic-acid modified agents ((99m)Tc-CSFA and (99m)TcN-CSFADTC) had obviously higher uptake in FR-positive kidney and much lower liver and spleen uptakes than that of non-folic-acid modified (99m)Tc-agent, and the kidney uptakes of FA-modified agents could be blocked significantly by the corresponding cold ligand. Furthermore in vitro and in vivo specific studies will be done in cell line and tumor bearing mice to confirm the usefulness of this chitosan backbone for FR targeting agent design.     

A new method for labelling of monoclonal antibodies and their fragments with technetium-99m

A new method of labelling of antibodies with technetium-99m is described. The antibody or fragment is derivatized by iminothiolane. Ligand exchange from a labile technetium complex such as TcO(GLU)₂⁻ results in >90% of the technetium labelling the antibody. This method appears to be equally applicable to antibodies and fragments.     

A propolis extract and the labeling of blood constituents with technetium-99m

Since ancient times propolis has been employed for many human purposes because to their favourable properties. Blood constituents labeled with technetium-99m (99mTc) have been used in nuclear medicine procedures. Some authors have reported that synthetic or natural drugs can interfere with the labeling of blood constituents with 99mTc. The aim of this work was to evaluate the action of a propolis extract on the labeling of blood elements with 99mTc. Samples of whole blood of male Wistar rats were incubated in sequence with an aqueous propolis extract at different concentrations, stannous chloride and 99mTc, as sodium pertechnetate. Blood samples were centrifuged to separate plasma and blood cells, soluble and insoluble fractions of plasma and blood cells were also separated after precipitation in trichloroacetic acid solution and centrifugation. The radioactivity was counted and the percentage of incorporated radioactivity (%ATI) for each fraction was calculated. The data obtained showed that the aqueous propolis extract used decreased significantly the %ATI in plasma proteins at higher concentration studied. Results suggest that at high concentration the constituents of this extract could alter the labeling of plasma proteins competing with same binding sites of the 99mTc on the plasma proteins or acting as antioxidant compounds.