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.     

Evaluation of the in vitro effect of a Lantana camara extract on the labeling of blood constituents of rats with technetium-99m

Blood constituents labeled with technetium-99m (99mTc) have been used in nuclear medicine procedures and drugs are capable to interfere on this labeling. Lantana camara (lantana) has medicinal properties and it has been used in folk medicine. The aim is to verify the effect of a lantana extract on the labeling of blood constituents with 99mTc. Blood of rats was incubated with extract, stannous chloride and 99mTc, as sodium pertechnetate. Plasma (P) and blood cells (BC) were isolated, also precipitated with trichloroacetic acid and soluble (SF) and insoluble fractions (IF) were separated. The % of radioactivity (%ATI) in these samples was calculated. Samples of labeled BC were washed and the %ATI maintained (%ATI-M) in the BC was determined. The results showed that lantana extract decreased significantly (p < 0.05) in the IF-P from 70.24 +/- 2.59 to 11.95 +/- 3.07. This effect was not observed in the BC and IF-BC. The BC-%ATI-M was significantly (p < 0.05) decreased in all concentrations tested when the BC was washed. This fact was not observed in the control. Substances present on the extract should have redoxi action decreasing the concentration of the stannous ion and this condition could justify the effect on the IF-P. The results about the BC-%ATI-M should indicate a possible effect on the transport of ions through the erythrocyte membrane.     

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.     

The use of technetium-99m as a radioisotopic label to assess cell-mediated immunity in vitro

We have developed a radioisotopic microassay of cell-mediated immunity employing target cells prelabeled with technetium-99m (^99mTc), a high specific activity metastable gamma emitter. Labeling kinetics, release and reutilization, subcellular localization, and effects of ^99mTc on DNA and protein synthesis have been investigated. Target cells were optimally labeled with 10 mCi of ^99mTc at 37 °C for 10 min. Cyclic freezing and thawing released less than 10% of total bound radioisotope. Spontaneous leakage of ^99mTc by monolayer cells was negligible over 48 hr and that which was released appeared to be nonreutilizable. Cell fractionation revealed that nuclear, mitochondrial, and microsomal fractions all were labeled with ^99mTc. The incorporation of ³H-thymidine and ³H-amino acids was not impaired in ^99mTc-labeled cells.The alloimmune reactivity of C57BL/6 mice which had received A/J skin allografts was studied by means of the ^99mTc microcytotoxicity assay. Cell-mediated immunity was clearly evident at 7 days postgrafting, peaked at 14 days, and had declined to background levels by 21 days. These findings correlated well with initial acceptance and ultimate rejection of the allografts. The rapid labeling time without dependence upon cell division for incorporation, high specific activity, low spontaneous release, and nonreutilizability are important advantages of ^99mTc over other radionuclides which have been employed in in vitro assays of cell-mediated immunity.     

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.     

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.     

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.     

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.     

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.     

Synthesis of a diaminedithiol bifunctional chelating agent for incorporation of technetium-99m into biomolecules

The synthesis of a bifunctional chelating agent (BCA), 1, based on the diaminedithiol (DADT) ligand system, is described. The six-step synthetic sequence has been accomplished in 16% overall yield, affording 1, which contains a thiolactone as a reactive moiety, which permits direct coupling to nucleophiles without the formation of byproducts. The reactivity of 1 toward benzylamine and subsequent labeling of the ligand with technetium-99m has been evaluated as a model for preparation of various bioconjugates. Both coupling and exchange labeling occur in high yield under mild conditions, and competition reactions with diethylenetriaminepentaacetic acid (DTPA) indicate the superior stability of the technetium-99m-DADT complex. Preparation of BCA 1 thus provides a new avenue into technetium-labeled radiopharmaceuticals.     

Evaluation of the cytotoxic and mutagenic potentiality of technetium-99m in Escherichi coli.

Since technetium-99m (99mTc) was introduced in medical research it has become one of the most employed radionuclides in nuclear medicine. 99mTc is ideal for routine use on the labeling of different radiopharmaceuticals due to its favorable characteristics. However, some biological effects have been described. These effects may be related to internal conversion electron and/or Auger electron emissions from 99mTc decay that present high linear energy transfer and can generate reactive oxygen species (ROS) in the medium. We evaluated in Escherichia coli K12S and Salmonella typhimurium TA102, both proficient in DNA repair, contribution of those decay emissions on the cytotoxicity induced by 99mTc, both either by generating lesions on DNA or by inducing alterations at membrane. We also studied the genotoxic and/or mutagenic potentiality of 99mTc, in Salmonella typhimurium, using the Ames test. The results showed that: i/ 99mTc is cytotoxic to the Escherichia coli K12S strains; ii/ this effect is related to the electrons (Auger and internal conversion) emissions, and iii/ the 99mTc is not mutagenic and/or genotoxic, when measured by Ames test.     

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.     

Synthesis and biological evaluation of a technetium-99m(I)-tricarbonyl-labelled phenyltropane derivative

A new tropane derivative was synthesized by combining a tridentate ligand, N-(2-picolylamine)-N-acetic acid (2-PAA), and a phenyltropane derivative. It was labelled with a [(99m)Tc(CO)(3)](+) moiety, resulting in the formation of two stable and neutral lipophilic isomers. Their identity was confirmed using radio-LC-MS. In normal mice, no brain uptake was observed for any of the isomers and in vitro autoradiography using mouse brain sections showed no specific uptake in the striatal area.     

Evaluation of the effect of mitomycin-C on the bioavailability of technetium-99m-labelled sodium pyrophosphate in mice.

We have reported that drugs alter the biodistribution of radiopharmaceuticals used in diagnostic imaging in nuclear medicine. Knowledge of such altered biodistribution is important in making diagnostic from scintigraphy. Mitomycin-C is used as component of many chemotherapeutic regimens to treat different tumors. The biological activities of mitomycin-C can be explained by its ability to inhibit deoxyribonucleic acid synthesis. Since patients on chemotherapeutic treatment can be submitted to nuclear medicine procedures, we studied the mitomycin-C effect on the bioavailability of the technetium-99m-labelled sodium pyrophosphate (9mTc-PYP) using an animal model. Mitomycin (0.45 mg) was administered by ocular plexus way Balb/c mice. One hour after the last dose, 99mTc-PYP (7.4 MBq) was administered and after 0.5 hr the animals (n = 15) were rapidly sacrificed. The organs were isolated, the radioactivity counted in a well counter and the percentage of radioactivity (%ATI) calculated. The results have shown that in the treated animals the %ATI has been decreased in spleen, thymus, heart and brain and increased in lung, liver and bone. The effect of this chemotherapeutic drug on the 99mTc-PYP biodistribution was statistically significant (Wilcoxon test, p < 0.05) and it could be explained by the metabolization or therapeutic action of mitomycin-C.     

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.     

Radiolabeling of HYNIC-annexin V with technetium-99m for in vivo imaging of apoptosis

Apoptosis is a critical factor in AIDS and other viral illnesses, cerebral and myocardial ischemia, autoimmune and neurodegenerative states, organ and bone marrow transplant rejection, and tumor response to chemotherapy and radiation. Improved methods to identify sites of apoptosis are increasing our understanding of the pathophysiology and treatment of these and numerous other human disorders. Here we describe the most used method for labeling annexin V, a protein with a high affinity for apoptotic cells in vitro, with technetium-99m (99mTc) as a radionuclide imaging agent that can localize and non-invasively quantify apoptosis in vivo when coupled with single-photon emission tomography. In this method, annexin V is first attached to the bifunctional chelator molecule hydrazino nicotinate (HYNIC). Once prepared, HYNIC-annexin V can be labeled with 99mTc, a widely available gamma-radiation-emitting radionuclide, for intravenous injection in as little as 30 min without the need for specialized reagents or equipment.     

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.