Studies of 99mTc-acylplasmins as agents for thrombus detection

It has been proposed that acylation at the active site of plasmin is able to prevent its reaction with 2-antiplasmin without affecting the fibrin affinity of the enzyme. To investigate the possibility that ^99mTc-labelled acylplasmins are improved thrombus-detecting agents, six acylating agents were synthesised and their reaction with plasmin and the labelling of the products with ^99mTc studies. Uptake of ^99mTc-acylplasmins in an in vitro thrombus model was complicated by precipitation processes, which may in part account for the rapid blood clearance in rabbits and high liver uptake in mice injected with the compounds. Quantitative measurements using an in vivo rabbit thrombus model demonstrated that guanidinobenzoyl-plasmin exhibited nearly a threefold increase in thrombus uptake compared with non-acylated ^99mTc-plasmin. The observed uptake is less than that obtained with ¹²⁵I-fibrinogen at clinically useful time intervals post-injection but represents a significant advantage over the use of ^99mTc-plasmin.     

Radiopharmaceuticals for thrombus detection

Most of the components of the thrombotic and fibrinolytic systems have at some time been evaluated as a means of carrying a radiolabel specifically to thrombi, although very few have been promising enough to emerge from investigational status to routine clinical use. New approaches are being explored, including improved methods of labeling platelets, chemically modified forms of previously tested plasma proteins, and new biomolecules, including monoclonal antibodies specific for fibrin and platelets. The current goal is to find one or more radiotracers that bind specifically and rapidly to thrombi, and that also have a rapid blood disappearance rate, permitting a clear diagnosis within a few hours after injection. Because this test may be needed to assess the course of therapy in an anticoagulated patient, the ideal radiopharmaceutical should be able to locate thrombi without interference by anticoagulants. Until a suitable thrombus-specific radiopharmaceutical becomes generally available, many hospitals will continue to attempt to make a diagnosis with nonspecific radiopharmaceuticals that can at best provide blood pool images to indicate filling defects. Several of the new approaches seem likely to provide the radiopharmaceutical sought, although clinical trials are at an early stage.     

Evaluation of radiocolloids as thrombus imaging agents. Effect of particle size on thrombus uptake

Thrombus uptake values of several 99mTc labeled radiocolloids determined using an experimental rodent model of deep venous thrombosis were correlated with particle size distributions. The thrombus uptake values increased with increasing mean particle size. The 99mTc-tin colloid had the highest thrombus uptake value of any of the colloids used in this study.     

Evaluation of DNA aptamers directed to thrombin as potential thrombus imaging agents

Two DNA aptamers directed against two separate exosites on human alpha-thrombin were evaluated for thrombus-imaging potential. Aptamer ODN 1 is directed to the thrombin substrate binding site (exosite 1). Our finding that ODN 1 competes with fibrin for binding to exosite 1 on thrombin suggests that ODN 1 will not be useful for thrombus imaging. Aptamer ODN 2 is directed against the thrombin heparin binding site (exosite 2). ODN 2 bound to model thrombi that were formed either by clotting purified fibrinogen with thrombin, or by recalcifying citrated plasma. As the thrombin content of thrombi was increased the rate of ODN 2 uptake into preformed thrombi increased, whereas the rate of release of ODN 2 out of preformed thrombi decreased. This in vitro data suggested that ODN 2 might be useful for thrombus imaging because it can bind to exosite 2 on fibrin-bound thrombin. However, in a rabbit jugular vein model using thrombus supplemented with human thrombin, ODN 2 uptake was equal to the ovalbumin control, and did not reflect thrombin content. While the in vitro results with ODN 2 were consistent with thrombus imaging, the rapid clearance of ODN 2 from circulation, combined with slow mass transfer in the clot, seem to work against in vivo thrombin-dependent imaging or washout analysis.     

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.     

Evaluation of six new 99mTc-IDA agents for hepatobiliary imaging

IDA derivatives of three substituted benzothiazol, and two substituted chlorophenyl and one substituted pyrazoline compounds have been labeled with 99mTc and screened with four rat models with hepatocellular dysfunction manifesting varying degrees of change of liver architecture and hepatocellular damage associated with an active parenchymal destruction, fatty metamorphosis and cirrhosis. Organ distribution studies at 1 h postinjection have been compared in normal and diseased animal models for each agent labeled with 99mTc and with 99mTc-Disofenin (Disida) and Lidofenin (Hida) and 131I-Rose Bengal. From the data obtained with the six new IDA derivatives, the distribution kinetics of 99mTc-Arclophenin, (N-N'-2-benzoyl-4-chlorophenyl)carbamoylmethyl) imino diacetic acid (Phenida), are closely comparable to 99mTc-Disofenin in all animal models. Crossover patient studies (n = 14) for clinical evaluation of 99mTc-Arclophenin vs 99mTc-Disofenin indicate the close similarity of the 2 agents with regard to blood pool retention, gross liver/heart ratios and liver washout, suggesting Arclofenin as a suitable agent for hepatobiliary function studies. The impaired hepatocellular animal models presented should serve for fast screening of hepatobiliary agents and enable comparison of a series of closely related compounds.     

A clinical comparison of 99mTc-DPD and two 99mTc-MDP agents

European Journal of Nuclear Medicine and Molecular Imaging - Two hundred and fifty-seven patients were studied with bone-seeking radiopharmaceuticals. They were randomly divided into three groups....     

Animal evaluation of technetium-99m triamide mercaptide complexes as potential renal imaging agents

Technetium-99m mercaptoacetylglycylglycylglycine (MAG3), a [99mTc]triamide mercaptide (N3S) compound has been synthesized in an attempt to obviate the stereochemistry problems associated with the diamide dimercaptide (N2S2) ligands. Because initial studies have been promising, the terminal glycine on the MAG3 compound has been varied to create a new series of N3S compounds. Twelve new N3S complexes were initially screened in mice and the more promising complexes, 99mTc mercaptoacetylgylcylglycyl-glycine [( 99mTc]MAG3), 99mTc mercaptoacetylgylcylglycyl-L-alanine [( 99mTc]MAG2-Ala), and both complexes of 99mTc mercaptoeacetylglycylglycyl-L-asparagine [( 99mTc]MAG2-Asn) and 99mTc mercaptoacetylglycylglycyl-L-glutamine [( 99mTc]MAG2-Gln), were further evaluated in rats utilizing constant infusion blood clearances, extraction efficiencies and protein binding assays. The renal excretion of all these complexes compared favorably with simultaneously administered [131I]OIH and [125I]iothalamate. The triamide mercaptide complexes represent a new ligand class for 99mTc, which may provide a variety of complexes for the evaluation of renal tubular function.     

Improved molecular imaging contrast agent for detection of human thrombus.

Molecular imaging of microthrombus within fissures of unstable atherosclerotic plaques requires sensitive detection with a thrombus-specific agent. Effective molecular imaging has been previously demonstrated with fibrin-targeted Gd-DTPA-bis-oleate (BOA) nanoparticles. In this study, the relaxivity of an improved fibrin-targeted paramagnetic formulation, Gd-DTPA-phosphatidylethanolamine (PE), was compared with Gd-DTPA-BOA at 0.05-4.7 T. Ion- and particle-based r(1) relaxivities (1.5 T) for Gd-DTPA-PE (33.7 (s*mM)(-1) and 2.48 x 10(6) (s*mM)(-1), respectively) were about twofold higher than for Gd-DTPA-BOA, perhaps due to faster water exchange with surface gadolinium. Gd-DTPA-PE nanoparticles bound to thrombus surfaces via anti-fibrin antibodies (1H10) induced 72% +/- 5% higher change in R(1) values at 1.5 T (deltaR(1) = 0.77 +/- 0.02 1/s) relative to Gd-DTPA-BOA (deltaR(1) = 0.45 +/- 0.02 1/s). These studies demonstrate marked improvement in a fibrin-specific molecular imaging agent that might allow sensitive, early detection of vascular microthrombi, the antecedent to stroke and heart attack.     

Preparation and preliminary evaluation of technetium-99m-labeled fragment E1 for thrombus imaging.

Fragment E1 labeled with 123I has been previously shown to permit imaging of thrombi in patients within as little as 20 min after injection. Because of the relatively rapid localization and blood disappearance of this protein, 99mTc would be the most clinically acceptable radionuclide for labeling Fragment E1. In this study, human fragment E1 was derivatized with a hydrazino nicotinate function to permit radiolabeling with reduced technetium. The modification reaction was carried out while the fragment E1 was protected in a complex, so that the modification occurred in nonfunctional regions of the fragment E1 molecule. After radiolabeling with 99mTc, the modified fragment E1 retained its functional activity, as judged by its binding to fragment DD in vitro. The ability of 99mTc-fragment E1 to produce images of venous thrombi was demonstrated in animal models. Images were focally positive within 20 min to 1 hr after injection. Thrombus-to-blood ratios exceeded those from 125I-fibrinogen in the same animals. This method of labeling appears to provide an alternative radiolabel to 123I without compromising the function of fragment E1.     

The influence of reducing agents on the composition of 99Tc-complexes: implications for 99mTc-radiopharmaceutical preparation

The use of formamidine sulphinic acid as a reducing agent in the presence of technetium-99-pertechnetate and diethyldithiocarbamate ligand has been shown to yield a complex containing a Tc = CO bond. The carbon monoxide present in this complex originates from the reducing agent itself. Evidence is presented which suggests that this carbonyl complex is present in the 99mTc-diethyldithiocarbamate preparation obtained by the use of formamidine sulphinic acid as the reducing agent. Use of hydrazine as a reducing agent yields a complex containing a Tc = N bond. It is apparent that when reducing agents such as hydrazine or formamidine sulphinic acid are used in the preparation of 99mTc-radiopharmaceuticals, the possibility of the formation of complexes structurally different to those obtained by the use of stannous chloride must be considered.     

Synthesis of technetium-99m labeled diaminodithiol for bifunctional chelating agents

Diaminodisulfide analogs (2a–b) were prepared by reduction of the intermediate Schiff's bases (1a–b) with sodium cyanoborohydride (NaBH₃CN) in dry diethyl ether under acidic condition without inducing internal cyclization reaction. The less sterically hindered NH proton of the diaminodisulfide (2b) could be successfully displaced by functional groups. The diaminodulfides (2a–b, 4c) were quickly converted to diaminodithiol (DADT; 5a–b, 6c) by reduction with a small amount of lithium aluminium in refluxing dry diethyl ether for 20 min, whereas reduction of bicyclic diaminodisulfides (3a–b) with excess LiAlH₄ did not produce the desired DADT (5a–b). Reduction of the other bicyclic diaminodisulfide including a functional group (methyl group) (3′c) with excess LiAlH₄ produced the DADT including a functional group in the sterically hindered nitrogen atom (8). Diaminodiathiol analogs including an N-functional group, such as N-p-iodophenethyl (6d), were synthesized without deiodination by a new revised procedure. The chelating ability of these ligands (5a–b, 6c–d, 8c) for Tc-99m was determined by thin-layer chromatography (TLC). The radiochemical yield of the Tc-99m labeled 5a–b, 6c–d, 8c was more than 95%. These results indicated that the DADTs including a functional group in the less sterically and the sterically nitrogen atom prepared by our new method have high chelating ability and high stability of Tc-99m and appear to be an attractive candidate as a useful chelator for bifunctional chelating agents.     

The influence of reducing agents on the composition of 99Tc-complexes: Implications for 99mTc-radiopharmaceutical preparation

The use of formamidine sulphinic acid as a reducing agent in the presence of technetium-99-pertechnetate and diethyldithiocarbamate ligand has been shown to yield a complex containing a Tc=CO bond. The carbon monoxide present in this complex originates from the reducing agent itself. Evidence is presented which suggests that this carbonyl complex is present in the ^99mTc-diethyldithiocarbamate preparation obtained by the use of formamidine sulphinic acid as the reducing agent. Use of hydrazine as a reducing agent yields a complex containing a Tc N bond. It is apparent that when reducing agents such as hydrazine or formamidine sulphinic acid are used in the preparation of ^99mTc-radiopharmaceuticals, the possibility of the formation of complexes structurally different to those obtained by the use of stannous chloride must be considered.