Evaluation of formamidine sulfinic acid and other reducing agents for use in the preparation of Tc-99m labeled radiopharmaceuticals.

Various reducing agents have been evaluated for their potential usefulness in the preparation of 99mTc labeled radiopharmaceuticals for use in nuclear medicine. Adequate labeling of various radiopharmaceuticals was accomplished using formamidine sulfinic acid. Nitrogen-purging of solutions is not required, which is an advantage for in-house preparation. Tagging requires heating, however, so heat-labile material cannot be used. Various compounds that could not be labeled when stannous chloride was used, could be tagged with 99mTc when formanidine sulfinic acid was used as the reducing agent.     

Preincubation with Sn-complexes causes intensive intracellular retention of 99mTc in thyroid cells in vitro

Technetium radiopharmaceuticals are well established in nuclear medicine. Besides its well-known gamma radiation, 99mTc emits an average of five Auger and internal conversion electrons per decay. The biological toxicity of these low-energy, high-LET (linear energy transfer) emissions is a controversial subject. One aim of this study was to estimate in a cell model how much 99mTc can be present in exposed cells and which radiobiological effects could be estimated in 99mTc-overloaded cells. Methods: Sodium iodine symporter (NIS)-positive thyroid cells were used. 99mTc-uptake studies were performed after preincubation with a non-radioactive (cold) stannous pyrophosphate kit solution or as a standard 99mTc pyrophosphate kit preparation or with pure pertechnetate solution. Survival curves were analyzed from colony-forming assays. Results: Preincubation with stannous complexes causes irreversible intracellular radioactivity retention of 99mTc and is followed by further pertechnetate influx to an unexpectedly high 99mTc level. The uptake of 99mTc pertechnetate in NIS-positive cells can be modified using stannous pyrophosphate from 3-5% to >80%. The maximum possible cellular uptake of 99mTc was 90Bq/cell. Compared with nearly pure extracellular irradiation from routine 99mTc complexes, cell survival was reduced by 3-4 orders of magnitude after preincubation with stannous pyrophosphate. Conclusions: Intracellular 99mTc retention is related to reduced survival, which is most likely mediated by the emission of low-energy electrons. Our findings show that the described experiments constitute a simple and useful in vitro model for radiobiological investigations in a cell model.     

Pharmacodynamics of stannous chelates administered with 99mTc-labeled chelates.

The pharmacodynamics of several tin compounds were studied in healthy rabbits, rabbits with myocardial infarcts, and isolated myocardial tissue. The results showed that tin chelates of pyrophosphate, HEDP, DTPA, and glucoheptonate are very unstable in vivo, giving rise to free stannous ions. These ions localize mainly in bone, with the rest being primarily excreted in the urine. They also concentrate more in infarcted than in normal myocardium; there they enter the mitochondria. The supernatant of homogenates contains bound and free fractions, demonstrating a subcellular distribution pattern similar to that of calcium ions. Tin chelates have different pharmacodynamics from the corresponding 99mTc chelates.     

An evaluation of six kits of technetium 99m human serum albumin injection for cardiac blood pool imaging.

We have investigated the suitability of five different commercially available kits which provide human serum albumin (HSA) labelled with technetium 99m (99mTc) for cardiac blood pool imaging. Four of these products were one-step processes using stannous chloride as the reducing agent; the fifth was based on an electrolytic reduction. In addition, we also assessed our own modification of the electrolytic method. We measured the radiochemical purity by precipitation with trichloroacetic acid and by gel filtration on a Biogel P4 column. In addition, we measured the clearance of radioactivity from the blood at frequent time intervals after intravenous injection. Each product was assessed in separate groups of six patients. The labelling efficiency of the one-step kits varied between 73 and 93% compared with 94 and 98% for the electrolytically labelled albumin. The blood clearance for all one-step kits was significantly faster than that obtained for the radiopharmaceuticals prepared by the electrolytic method. We conclude that HSA labelled with 99mTc by the electrolytic method is to be preferred.     

Kinetic analysis of technetium-99m d,1-HM-PAO decomposition in aqueous media

Technetium-99m-d,1-hexamethylpropyleneamine oxime [99mTc]d,1-HM-PAO) is a neutral-lipophilic chelate formed from a kit in high yield by stannous reduction of Na99mTcO4-. Of three methods used to analyze the purity of the 99mTc complexes, a single strip method using ether as the developing solvent was the most rapid and simple to perform. The lipophilic chelate converts to 99mTcO4- and other hydrophilic products with time and this limits the useful lifetime of the preparations. The rate of decomposition of [99mTc]d,1-HM-PAO increased in the presence of excess stannous ion and at pH greater than 9.     

Technetium-99m labeling of red blood cells: in vitro evaluation of a new approach

By titration of two different stannous kits for 99mTc labeling of red blood cells (RBC) we found concentrations of 1-2 micrograms tin per ml of blood to give the highest labeling yield. Using a new kit containing 2 micrograms of tin and 0.1% hypochlorite (NaOCl) as an oxidizing agent we labeled RBC with 99mTc avoiding centrifugation of cells. To evaluate this new procedure we assessed the dependency of tin incubation time, and addition of 4.4% EDTA as a chelating agent on labeling efficiency. We also measured the dependency of EDTA on the stability of the label. Optimal conditions for labeling of 1 ml of whole blood using the new stannous kit were: 5-10 min of tin incubation, 0.2 ml of 0.1% hypochlorite, and 15 min of 99mTc incubation. This procedure resulted in a labeling efficiency of at least 96%. The overall effect of EDTA was not an increased labeling efficiency, and EDTA increased the stability of the label with only a few percent. The promising results of this new labeling approach encourage to further laboratory investigations and eventual clinical evaluation of the procedure.     

Imaging of abdominal infection using 99m Tc stannous fluoride colloid labelled leukocytes

Radiolabelling of leukocytes using labelled phagocytosed technetium-99m (99mTc) colloidal radiopharmaceuticals has been reported as a method for imaging infection. This in vivo study compares the use of leukocytes labelled using 99mTc stannous fluoride colloid with leukocytes labelled using indium-111 (111In) oxinate. A total of 26 patients (10 male, 16 female; mean age 52 years, range 23-88 years) referred for the investigation of possible infection were studied using both leukocyte labelling methods simultaneously. Images were acquired 4h and 24h after re-injection of the labelled cells. The images were evaluated qualitatively by two nuclear medicine physicians. The results show a high degree of concordance between the techniques: 11 of the 28 images showed a focus of leukocyte accumulation with both techniques at 24h, and 13 out of 28 showed a normal appearance at 24h with both methods. In four cases the results were discordant; the 99mTc stannous fluoride colloid labelled leukocytes gave a false positive appearance at 24h in three patients and a false negative in one. In conclusion, colloid labelling of leukocytes offers a sensitive method for the detection of infective foci coupled with the high resolution imaging offered by 99mTc. It has the advantage over other in vitro labelling methods of being a simpler, non-labour-intensive procedure employing whole blood, and its use should be considered by departments that have limited facilities for in vitro leukocyte labelling.     

New preparation method for 99mTc-phytate.

A new method is described in which Pt-Sn or Sn-Sn electrodes are used to activate 99mTcO4-. The 99mTc is incorporated into phytic acid by stannous ion released from a tin anode by the corrosive reaction. The most suitable pH for labeling phytic acid by this method was below 5 and the 99mTc-phytate formed could be precipitatedd with Ca+2 at a pH above 3.5. Though 99mTc-phytate is soluble in aqueous solution, it forms an insoluble species with Ca+2 in vivo and is trapped in the reticuloendothelial system. More than 93% of the 99mTc-phytate localized in the liver of mice; here its biologic half-time is about 112 hr. The 99mTc-phytate prepared by this method promises to be useful as a liver-scanning agent. Since our method does not require an applied electric potential, it appears to be one of the most convenient methods for labeling compounds with 99mTc.     

The integrity of the disulfide bond in a cyclic somatostatin analog during 99mTc complexation reactions

Recent development of a variety of thiol-free chelating agents has facilitated the design of 99mTc-labeled somatostatin analogs suitable for receptor imaging of somatostatin-positive tumors. However, it remains ambiguous whether the disulfide bonds in cyclic peptides are stable during 99mTc complexation reactions, and contradictory results have been reported regarding the integrity of disulfide bonds in cyclic somatostatin analogs. To estimate the stability of the disulfide bond in a synthetic somatostatin analog at low peptide concentrations, [125I]I-RC-160, in which radioiodine was incorporated into the 3-Tyr residue, was synthesized and the integrity of the disulfide bond of the peptide was investigated in the presence of reducing agents such as ascorbic acid, dithionite, and stannous ions. The disulfide bond in [125I]I-RC-160 remained stable in the presence of ascorbic acid in boiling water. The disulfide bond was also stable when treated with stannous ions at concentrations sufficient to reduce 99mTc for complexation with a thiol-free chelating agent, bis(hydroxamamide) analog when the 99mTc complexation reaction was performed at room temperature. However, the disulfide bond of [125I]I-RC-160 was slightly cleaved in the presence of a small amount of stannous ions when the reaction was performed in boiling water. Treatment of [125I]I-RC-160 with dithionite in boiling water markedly reduced the disulfide bond of the parental peptide. These findings indicated that synthetic somatostatin analogs may be labeled with 99mTc with stannous ions as the reducing agent without impairing their structure after conjugation of thiol-free chelating agents that provide 99mTc chelates under mild reaction conditions.     

Technetium-99m-labeled phosphonic acid analog of serine: bone uptake

A phosphonic acid analog of serine (PAAS), its P-methyl derivative, and an allied compound were labeled with 99mTc, and evaluated in experimental animals. These molecules were able to bind 99mTc in the presence of stannous ions, but the biologic behavior of the later two labeled compounds was quite different from that of the first one. Technetium-99m-labeled PAAS behaved like 99mTc-labeled methylene diphosphonate, but its P-methyl derivative and the third agent showed little bone accumulation. It appears that both hydroxyl groups attached to the phosphorus atom are essential for uptake in bone, but that only one is required for binding 99mTc.     

Organ distribution of 99m-Tc-and 51-Cr-labelled thymocytes.

We have employed 99mTc as a radioisotopic label to study the organ distribution of murine thymocytes. The compartmentalization of 99mTc-labeled cells that had not been reduced by treatment with stannous chloride was similar to that of 51Cr-labeled cells and was characterized initially by 48-50% uptake of the injected radioactivity by the lungs. Increased hepatic and splenic and decreased pulmonary localization were noted at 1 hr and these shifts were more pronounced by 4 hr. Technetium-99m-labeled cells reduced by stannous chloride had significantly different patterns of hepatic, pulmonary, and splenic localization and at 4 hr the lungs still retained 24% of the injected radioactivity compared with only 3% in the spleen. Size distribution studies revealed that unlabeled as well as unreduced and reduced 99mTc-labeled thymocytes were almost identical to one another so that differences in compartmentalization could not be attributed to this factor. Since reduction with stannous chloride did not alter the distribution of 51Cr-labeled cells, this suggested some type of complex interaction between stannous ions and the labeling species of 99mTc. The in vivo localization of intravenously administered Na99mTcO4 and Na2CrO4 was markedly different from the corresponding radiolabeled cells thereby indicating that the distribution patterns that we observed truly represented cell-associated radioactivity. Although it may not necessarily be the proper reference point, the similarity in organ distribution of 99mTc- and 51Cr-labeled cells should allow direct comparison of previously reported data employing this radionuclide and that obtained in future studies with 99mTc.     

Newer methods of labeling diagnostic agents with Tc-99m

The past several years have seen marked advances in technetium/rhenium chemistry applicable to the preparation of new 99mTc-labeled radiopharmaceuticals. This article focuses on recent developments in technetium chemistry, including the preparation of "3 + 1" complexes, the preparation and use of (99mTc[CO]3)+ complexes for labeling biomolecules, the preparation of rhenium steroid inclusion complexes, improvements in both hydrazinonicotinamide labeling chemistry and in the preformed 99mTc complex method of labeling biomolecules, and new solid-phase separation techniques that may allow the isolation of high specific-activity radiopharmaceuticals in a clinical setting.     

The preparation of dry, monodisperse microspheres of [99mTc]albumin for lung ventilation imaging

2 micron microspheres of albumin were prepared and labelled with 99mTc according to published methods. However, when the labelling technique was repeated without the inclusion of microspheres, apparent labelling, which was attributed to the formation of a 99mTc tin colloid, was still observed. It was not possible to identify 99mTc tin colloid in the presence of 99mTc microspheres. To establish conditions under which there was no 99mTc tin colloid formation, labelling, with and without microspheres, was studied at low pH for a range of SnCl2 X 2H2O concentrations. A "kit" containing 0.4 mg of microspheres and 1 microgram of SnCl2 X 2H2O in 1 ml of 25 mM HCl gave optimum labelling of 88.3% (SD 3.9, n = 38). Unlabelled and labelled kits were stable for 1 and 3 h respectively. Before administration, the microspheres were resuspended in ethanol, in which they were stable for 6 h. None of the preparations contained 99mTc tin colloid.     

Microautoradiographic study of technetium-99m colloid uptake by the rat liver

A new microautoradiographic technique was developed to study the distribution of 99mTc-labeled radiopharmaceuticals. Using a thick emulsion, it is possible to get microscopically visible tracks of internal conversion and Auger electrons. The liver uptake of microscopic particles has been thought to occur in Kupffer cells but no direct evidence has been provided for technetium colloids. Using this method, 99mTc-labeled colloids were clearly identified in Kupffer cells in the sinusoidal areas of liver. "Track" microautoradiography using a thick emulsion layer may be used on any frozen tissue sections and may provide an important tool to assess the biodistribution of 99mTc radiopharmaceuticals.     

99mTc-ENS: a new radiopharmaceutical for aerosol lung scintigraphy. Comparison between different freeze-dried formulations.

Exogenous natural surfactant (ENS) labeled with 99mTc(99mTc-ENS) is a new radiopharmaceutical for pulmonary aerosol scintigraphy. In this study, different freeze-dried formulations were evaluated to develop a suitable and long-storage method for the ENS, the nonradioactive precursor of this radiopharmaceutical. METHODS: Two freeze-dried formulations were evaluated: the sterile ENS suspension-stannous chloride altogether lyophilized (chlorlioENS) and the lyophilized sterile ENS suspension with the addition of stannous chloride as a solid drug (lioENS). These precursors were stored at room temperature for 3 mo and then labeled with 99mTc. For comparative purposes, the sterile ENS suspension with the addition of stannous chloride labeled with 99mTc(99mTc-chlorENS) was also studied. The quality controls for each radiopharmaceutical were performed by an ascending paper chromatography to determine the labeling yield percentages. The study was performed in 30 female Sprague Dawley rats, which inhaled each radiopharmaceutical by nebulization. Twenty-five minutes after the aerosol inhalation, the animals were killed to extract their organs and measure their activity in a gamma spectrometer. The data are given as the percentage of activity concentration (C%) for each organ. RESULTS: The physicochemical properties of lioENS were adequate for a freeze-dried product. The labeling yields for 99mTc-lioENS and for 99mTc-chlorENS were always greater than 95% even after nebulization. The results of the biologic distribution studies showed that the activity concentration found in lungs for these radiopharmaceuticals were 95.7% +/- 2.6% and 96.7% +/- 2.6% respectively, results that do not differ statistically. On the other hand, the activity concentration found in lungs for the 99mTc-chlorlioENS (31.3% +/- 11.1%) and its labeling yield percentages (<10%) are statistically different (P < 0.05) from the results obtained with the two radiopharmaceuticals mentioned above. CONCLUSION: Taking into account the lioENS physicochemical properties, its long shelf life and that 99mTc-lioENS shows the same radiochemical and radiopharmacological behavior of the 99mTc-chlorENS, it can be concluded that the 99mTc-lioENS can be used for aerosol lung scintigraphy.     

Mechanistic aspects of the interaction of 99mTc in association with stannous pyrophosphate with damaged red blood cells

The binding of the various components of [99mTc] stannous pyrophosphate and stannous pyrophosphate/pertechnetate to damaged red blood cells is studied. It is shown that the pyrophosphate molecule enters the damaged red blood cells when the pyrophosphate concentration in blood is greater than 100 nmol/ml but does so as the uncomplexed ion. Uptake of 99mTc when introduced as [99mTc]stannous pyrophosphate is constant at approximately 18%. If the 99mTc is introduced as pertechnetate after the damaged cells are mixed with stannous pyrophosphate then at low stannous ion concentrations the uptake is directly dependent on the stannous ion concentration. However, at higher stannous ion concentrations the uptake of technetium by the damaged red cells decreases, but this decrease appears to result from several independent aspects of the sample, such as the binding of the technetium to the plasma proteins and the displacement of the technetium by pyrophosphate within the damaged cell.     

Studies on the labeling of streptokinase with 99mTc for use as a radiopharmaceutical in the detection of deep-vein thrombosis: concise communication.

Streptokinase was labeled with 99mTc using both stannous chloride and stannous pyrophosphate as reducing agents. Sixty to seventy-five percent of the 99m Tc was incorporated into streptokinase using stannous chloride as a reducing agent at pH 1-2, wheras 50-60% was incorporated using stannous pyrophosphate at neutral pH. Increasing the pH from 2 to 7 in the presence of stannous chloride caused the release of 15-20% of the protein-bound 99mTc. Incorporation of 99mTc into protein was relatively slow: labeling required 2-3 hr at room temperature. The concentration of stannous pyrophosphate required for optimum labeling varied between 10(-5) and 10(-2) M. Polyacrylamide-gel electrophoresis showed that the filler substance in commercial streptokinase was also labeled with 99mTc. However pure streptokinase gave a homogenous protein band after polyacrylamide-gel electrophoresis. This protein band coincided with the peak of streptokinase-bound 99mTc. The results obtained may partially explain why 99mTc-labeled streptokinase lacks the necessary specificity for the satisfactory location of blood clots in vivo.     

Technetium-99m labeling of murine monoclonal antibody fragments

F(ab')2 fragments of several murine monoclonal antibodies have been labeled with 99mTc by a direct, pretinning method. The fragments were incubated with stannous ions overnight to split disulfide groups--a process which converts dimeric F(ab')2 to monomeric fragments. The pretinned fragments were then either directly labeled with 99mTc, frozen for subsequent labeling, or lyophilized to make kits for 99mTc-labeling at some later date. The 99mTc-labeled fragments were shown to be stable against transchelation when challenged with ethylenediaminetetraacetic acid, retained immunoreactivity, and were capable of binding to human tumor xenografts in nude mice.     

Development and validation of hydroxy ethyl starch kits for instant use in gastroesophageal reflux and gastric motility studies

A reduction method based on stannous chloride is described to prepare hydroxy ethyl starch kits for gastrointestinal reflux and gastric motility studies. Following verification of the consistency of radiolabelling, in vitro experiments were carried out to validate 99mTc hydroxy ethyl starch as a liquid phase and solid phase gastric motility imaging radiotracer. Gastroesophageal reflux, liquid phase and solid phase studies were then conducted in 13 adult volunteers to examine the in vivo stability of the radiotracer. High labelling efficiency (>95% when prepared at neutral pH) was consistently achieved, which remained stable in conditions simulating gastric environment. Twelve of the 13 volunteers did not show absorption of any radioactivity from the gastro-intestinal tract. 99mTc hydroxy ethyl starch is a new agent suitable for gastroesophageal reflux and gastric motility studies. It is available in kit form and is a more 'physiological' agent than 99mTc sulphur colloid for preparing a solid radioactive meal. 99mTc hydroxy ethyl starch represents a true carbohydrate meal, and unlike 99mTc sulphur colloid, is easy to prepare and can easily be standardized to produce a standard vegetarian meal.