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.     

Synthesis and biodistribution studies of technetium-99m-labeled aminopeptidase N inhibitor conjugates

Probestin is a potent aminopeptidase N (APN) inhibitor. Four probestin conjugates containing a tripeptide chelator (N₃S) and a PEG₂ linker were synthesized and radiolabeled with Tc-99m. The number of –COOH groups on the chelator was altered to increase the excretion of the radiotracer from blood stream via the renal-urinary pathway and to decrease its hepatobiliary uptake. Biodistribution of the radiolabeled conjugates was evaluated in healthy CF-1? mice at 1 h post-injection. The results revealed that the Tc-99m labeled probestin conjugate preferentially (>85% injected dose) excreted via the renal route when an aspartic acid residue was added to the linker (conjugate 4). These results suggest that the pharmacokinetic properties of probestin-based APN-targeted agents could be optimized by adding an appropriate amino acid residue in between the linker and the payload.     

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.     

Convenient preparation of no-carrier-added technetium-99m radiopharmaceuticals using solid-phase technology.

A solid-phase technetium chelation chemistry was developed as a means of preparing (99m)Tc radiopharmaceuticals at high effective specific activity (HSA). Three peptidic N(3)S (99m)Tc ligands [mercaptoacetyl-Gly-Gly-Gly (MAG3), picolinyl-Ser-Cys-Gly-Thr-Lys-Pro-Pro-Arg (RP063), and dimethyl-Gly-Ser-Cys-Gly-Thr-Lys-Pro-Pro-Arg (RP128)] were used. The free thiol of Cys in each was attached to a series of commercially available amine-functionalized supports in a two-step process. The amine groups on the solid supports were converted to maleimide groups followed by the attachment of the (99m)Tc chelators through a thiol ether linkage with Cys. The optimized loading of the supports ranged 6-122 micromol/g support as determined by amino acid analysis. Each of the peptide-loaded supports (50-100 mg) was placed in either glass syringe vessels or disposable chromatography columns. Labeling with [(99m)Tc]pertechnetate (200-800 MBq) in the presence of stannous gluconate was achieved at room temperature for 30-60 min or in a 100 degrees C water bath for 10 min. Up to 80% of the activity was eluted from the column with saline to give products with purity up to 99.8% as determined by HPLC. Amino acid analysis indicated as little as 100 pmol of peptide present in the (99m)Tc products, demonstrating that extremely high effective specific activity can be achieved without the need for purification.     

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.     

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.     

Imidazoles as well as thiolates in proteins bind technetium-99m.

99mTc is widely thought to directly bind proteins through thiolate groups of cysteine residues, resulting in Tc-cysteinyl-protein bonds. Chemical reduction of disulfide bonds in proteins is widely used to generate thiolates with the goal of increasing 99mTc binding. This strategy is used because most proteins contain no thiolates, but many do contain disulfide bonds. In this study, we have evaluated the hypothesis that imidazole groups of histidine are also involved in direct 99mTc binding to proteins. Human gamma-globulin was used as the model protein in these studies. The immunoglobulin was used (a) without reduction or was (b) treated with stannous ions to reduce disulfide bonds thereby increasing thiolate concentration. These proteins were used to evaluate the hypothesis that imidazole as well as thiolate groups bind Tc. The proteins were evaluated by (a) using free amino acids to compete with proteins for 99mTc and (b) by chemical modification of amino acid side chains. In addition, peptides known to contain either cysteine or histidine, but not both, were also successfully directly labeled with 99mTc. These results indicate that in proteins (and peptides) imidazole-containing groups as well as thiolate-containing groups bind Tc.     

An N2S2 bifunctional chelator for technetium-99m and rhenium: complexation, conjugation, and epimerization to a single isomer.

A bifunctional chelator 6 was prepared bearing an N2S2 core for binding rhenium or technetium and a carboxylic acid group for conjugation to amino groups of biomolecules. Complexation of 6 with rhenium(V) resulted in two kinetic isomers, anti-7 and syn-7, being formed in approximately equal amounts. Epimerization with 0.5 M NaOH yields a single isomer anti-7, as determined by NMR spectroscopy and single-crystal X-ray analysis. The 99mTc complex was prepared at the tracer level by reaction of the ligand with 99mTcO4-, tin(II) chloride and sodium gluconate giving a mixture of two isomers, but showing a preference for the anti isomer. Chelation in the presence of 1 M NaOH results in anti-8 being formed as the sole product. The bifunctional ability of the ligand was explored by amide formation with (S)-alpha-phenethylamine, either by direct DCC coupling or through the N-hydroxy succinimidyl ester 9 intermediate. The deprotected bioconjugate 11 was complexed with rhenium, yielding similar amounts of two isomeric rhenium complexes, anti-12 and syn-12, which were isolated and characterized by NMR spectroscopy. Treatment of the kinetic mixture of anti-12 and syn-12 with 1 M NaOH resulted in quantitative conversion to a single rhenium complex anti-12. With technetium-99m in 0.1 M sodium acetate, bioconjugate 11 yielded both technetium-99m complexes anti-13 and syn-13, in a 2:1 ratio, respectively. In contrast, complexation in the presence of 1 M NaOH gave only one technetium-99m complex, assigned the structure anti-13.     

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.     

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.     

Synthesis and biological properties of the lipophilic technetium-99m complex 99mTc(acac)3

In the development of technetium-99m radiopharmaceuticals for the evaluation of regional cerebral perfusion, one series of complexes that has remained unexplored is the neutral lipophilic tris complexes formed with β-diketonato ligands. The prototype complex of this series, tris(2,4-pentanedionato) technetium(III), has been prepared via a new synthetic route and chemically characterized using ⁹⁹Tc and the biodistribution of the no-carrier-added ^99mTc complex has been determined. The ^99mTc complex was found to be distributed throughout the body with persistant high blood levels indicative of a high degree of protein binding. The primary route of excretion was the hepatobiliary system as indicated by the appearance of ^99mTc in the gut and feces at longer sample times post-injection. Although this complex was not retained by the brain, it does provide a starting point from which a more effective agent might be developed.     

Influence of antipyretic drugs on the labeling of blood elements with technetium-99m

Acetaminophen (AAP), acetylsalicylic acid (ASA) and dipyrone (DIP) are antipyretic and analgesics drugs that have wide use in health sciences. Some drugs can modify the labeling of blood elements with technetium-99m (99mTc). This work has evaluated the effect of AAP, ASA and DIP on the labeling of the blood elements with 99mTc. Blood was incubated with different concentrations of the drugs before the 99mTc-labeled process. Plasma (P), blood cells (BC), insoluble (IF-P, IF-BC) and soluble (SF-P, SF-BC) fractions were separated and percentage of radioactivity (%ATI) in each fraction was determined. Data have shown that the antipyretic drugs used in this study did not significantly modify the fixation of 99mTc on the blood elements when the experiments were carried out with the doses usually used in human beings. Although the experiments were carried out with rats, it is possible to suggest that AAP, ASA or DIP should not interfere with the procedures in nuclear medicine involving the labeling of blood elements with 99mTc.     

Preparation of no-carrier-added technetium-99m complexes via metal-assisted cleavage from a solid phase

A novel method for the preparation of no-carrier-added (nca) complexes [99mTc(CO)3L] (L = diethylenetriamine or picolylamine-N-acetic acid) is described. The ligands were covalently bound to a solid support of organic polymers via formation of a tertiary amine from the chelating unit. This C-N bond to the solid phase is selectively cleaved during the formation of the technetium complexes by intramolecular nucleophilic attack of a remaining hydroxy ligand to the alpha-carbon. The complex [99mTc(CO)3L] is released into solution while uncomplexed ligand and uncleaved complex remain solid-phase bound. High specific activity technetium complexes can then be isolated by simple filtration. Cleavage yield depends on temperature, pH, and ligand. Up to 50% release from the solid phase could be achieved under optimized conditions. Corresponding to the 99mTc concentration, free ligand is present in concentrations lower than 10(-7) M. If a targeting vector is conjugated to these ligands, no-carrier-added radiopharmaceuticals can be prepared in that way.     

Trypanosoma cruzi: Biodistribution of technetium-99m pertechnetate in infected rats

With the aim of investigating the biodistribution of technetium-99 m pertechnetate () in rats infected with Y strain of Tripanosoma Cruzi, at the peak of parasitemia, (14th day of infection), we injected Wistar rats with 0.1 ml of (3.7 MBq). After 60 min, the percentage of radioactivity per gram was counted in several isolated organs and blood, using a gamma counter (1470 Wizard, PerkinElmer Finland). The uptake of increased significantly in blood and decreased in the colon of infected animals (p < 0.05). A significant reduction in serum iron and red blood cells and a significant increase in total proteins, leukocytes and lymphocytes in the infected rats were observed, compared with controls (p < 0.05). A reduction in muscle layer thickness of the colon and mononuclear inflammation were observed. These results conclusively demonstrate that T. cruzi infection would be associated with changes in the biodistribution of and in colon morphology, with potential clinical implications.     

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.