Technetium-99m ethylene dicysteine: a new renal tubular function agent

Technetium-99m ethylene dicysteine (EC), a metabolite of ethylene cysteine dimer (ECD), is a new technetium-labelled renal tubular function tracer introduced as an alternative to ortho-iodohippurate (OIH) and with imaging qualities similar to 99mTc-mercaptoacetyltriglycine (MAG3). The elimination of 99mTc-EC is principally via active tubular transport. It is available in lyophilised kit form which can be easily prepared at room temperature, and the compound remains stable for at least 8 h. Both in normal individuals and in patients, plasma clearance of 99mTc-EC has been reported to be around 0.75 of OIH clearance. Thus there is a very strict correlation between 99mTc-EC and OIH clearance, and several algorithms are available to estimate OIH clearance from 99mTc-EC clearance. The renal extraction ratio of 99mTc-EC is 0.70. The distribution volume of 99mTc-EC is twice that of 99mTc-MAG3 (20% of body weight) and slightly higher than that of OIH. The plasma protein-bound fraction of 99mTc-EC (30%) is significantly lower than that of 99mTc-MAG3 and OIH. The same applies to red blood cell binding of 99mTc-EC (5.7%). There is negligible uptake in the liver and intestines. Within 1 h 70% of 99mTc-EC is excreted in the urine. 99mTc-EC provides the same scintigraphic information as 99mTc-MAG3. The lower liver activity makes 99mTc-EC particularly attractive in patients with renal failure. The 99mTc-EC clearance can be accurately estimated from a single plasma sample obtained at 54 min after injection. In conclusion, 99mTc-EC is a suitable renal imaging agent and for some applications is even more attractive than OIH: it provides an index of tubular function and yields high-quality images. The labelling procedure is easy, radiochemical purity is high and the complex is stable for a long time. The extent to which 99mTc-EC is adopted for clinical use will ultimately depend upon its cost and availability.     

Pharmacokinetic characteristics of 99mTc-Ethylene-l-dicysteine (99mTc-EC)

BACKGROUND: Ethylenedicysteine-99mTc (99mTc-EC) has been more and more commonly applied in dynamic studies as well as for clearance determinations. However, it was necessary to investigate in detail the pharmacokinetic characteristics of the radiopharmaceutical which may be important for its applicability in assessment of renal function. RESULTS: Kidney images obtained from renoscintigraphy are characterised by excellent quality without visualisation of the organs adjacent to kidneys (liver, spleen). Renoscintigraphic curves demonstrate typical shapes with TMAX and T1/2 values not differing from the corresponding values obtained for other radiopharmaceuticals (99mTc-MAG3, 131I-OIH). In plasma, 99mTc-EC binds with proteins to a considerably lesser degree (c. 1/3) than 131I-OIH (c. 2/3), or 99mTc-MAG3 (> 9/10). No binding of 99mTc-EC with erythrocytes has been demonstrated, whereas 131I-OIH attaches to or penetrates the red blood cells (10-12%). 99mTc-EC is quickly excreted from the organism: 40 min after i.v. injection up to 70% of the administered radiopharmaceutical is found in urine, and at 1 and 1.5 h after the administration 80% and 95%, respectively. The distribution of 99mTc-EC in the organism can be described in a fully satisfactory way by means of an open two-compartment model, which allows this model to be used for clearance determinations. Comparison of the values of renal plasma clearance without collection of urine with the values determined by means of measurement of activity excreted with urine and mean blood concentration over a finite time interval leads to the conclusion that extrarenal plasma clearance of this compound (via the liver?) is negligible and amounts to c. 17 ml/min (5-6% of the total). The obtained correlation between clearance values for 99mTc-EC and 131I-OIH supports the contention that extrarenal excretion rate of 99mTc-EC (through the liver and bile ducts) is lower than the corresponding rates of either 131I-OIH or 99mTc-MAG3. A very close correlation between clearance values for 99mTc-EC and ERPF (131I-OIH clearance) and between their extraction constants (r = 0.91 and 0.92, respectively), allows for the introduction of 99mTc-EC to the assessment of renal function instead of 131I-OIH. Effective dose to the patient from unit activity of 99mTc-EC is comparable with that resulting from administration of other radiopharmaceuticals labelled with 99mTc.     

An investigation of factors that might influence the radiochemical purity and stability of 99mTc-MAG3

Technetium 99m mercaptoacetyltriglycine (99mTc-MAG3) was prepared from a commercial kit by various techniques to assess the effect of a number of variables on radiochemical purity and stability. Its radiochemical purity was determined by high performance liquid chromatography at 0 and 6 h after preparation and was found to be consistently greater than 95%. It has been demonstrated that the radiochemical purity of 99mTc-MAG3 prepared according to the manufacturer's instructions is not influenced by the volume of 99mTc generator eluate used, agitation, the presence of air in the reaction vial or the use of a 99mTc generator eluate with a 99Tc:99mTc ratio of 16:1. A modified method of preparation in which the MAG3 kit is reconstituted with saline before addition of 99mTc-pertechnetate has been shown to yield a satisfactory product and should help to minimise the radiation dose to the fingers of radiopharmacy staff.     

99mTc ethylene dicysteine scintigraphy for diagnosing cortical defects in acute pyelonephritis: a comparative study with 99mTc dimercaptosuccinic acid

AIM: To evaluate the clinical usefulness of early cortical phase images in the diagnosis of acute pyelonephritis and to compare the measurements of differential renal function obtained by both 99mTc ethylene dicysteine (99mTc-EC) and 99mTc dimercaptosuccinic acid (99mTc-DMSA). METHODS: Forty-three children who had undergone both 99mTc-EC and 99mTc-DMSA studies within 5 days of acute infection were studied. Cortical images of 99mTc-EC were obtained by the sum of the renogram frames achieved between the first 60-120 s of the renogram study. DMSA and EC images were visually interpreted using four points of semiquantitative ratings: 0, normal; 1, mild hypoactivity; 2, moderate hypoactivity with partial loss of margins; 3, marked hypoactivity with loss of cortical margins. Values for the differential renal function were obtained for both studies. RESULTS: DMSA detected a total of 109 lesions in 36 patients and EC detected 90 lesions with a sensitivity of 82.5%. 99mTc-EC successfully detected moderate-to-severe cortical lesions but was less effective with mild lesions (sensitivity 60%). Bland-Altman analysis demonstrated good agreement among the results for differential renal function (95% CI -0.26 to 0.96). CONCLUSION: 99mTc-EC early phase images detected most of the cortical lesions. Its sensitivity depends on the severity of the lesions. EC provided reliable information in the estimation of differential renal function.     

99mTc-labelled Cu(I)-EHDP, a potential skeletal imaging agent

99mTc-Cu-EHDP has been prepared with high labelling yield applying for the first time the method of instantly formed cuprous ions in the mixture. A gel chromatography column scanning technique has been used to study the 99mTc fractions in the preparation. The study of the influence of pH-value on the amount of 99mTc-CU-EHDP fraction shows that pH 1.6 - 1.7 gave the best labelling results. The formation rate of 99mTc-Cu-EHDP complex with a high labelling yield was fast and achieved within a few mins. This suggests the reduction of 99mTc-pertechnetate to Tc (IV). The final preparation was found stable for at least 4 hrs after mixing the reactants with the 99mTc-eluate. Comparative biokinetic studies of 99mTc-Cu-EHDP and 99mTc-Sn-EHDP in rabbits and mice showed a high bone uptake and fast elimination of 99mTc-Cu-EHDP from the skeleton. No significant difference was found in the plasma protein binding of 99mTc-Cu-EHDP and 99mTc-Sn-EHDP in rats as assessed by the GCS-technique. Radionuclide imaging in rabbits, using a gamma camera, showed 99mTc-Cu-EHDP to be a good bone-imaging agent.     

Electrophoretic analysis of different technetium-99m (SnCl2) methylene diphosphonate complexes

Technetium-99m MDP was prepared from MDP kits from several different sources. The resulting [99mTc]MDP preparations were analyzed by electrophoresis. The results demonstrated the presence of at least four different 99mTc complexes in these preparations. Modified kit preparations were analyzed to show the effects of concentration and pH on formation of the impurities. The electrophoresis results were correlated with scintillation camera imaging studies in rabbits and suggest that hydrolysis of MDP to phosphate and methylphosphate results in formation of 99mTc complexes with poor biological behavior as bone scanning agents.     

A novel 99mTc delivery system using (n,gamma)99Mo adsorbed on a large alumina column in tandem with Dowex-1 and AgCl columns.

99Mo of specific activity 7.4-14.8GBq (200-400mCi)/g was adsorbed on a 'jumbo' alumina column (40g, 90mm(H) x 20mm (dia.); 50g, 65mm(H) x 35mm(dia.). [99mTc]TcO4-, eluted with 50-70ml normal saline and applied to a tiny column of a strong anion exchanger, Dowex-1 x 8, 10-15 mg, 8-12 mm(H) x 1 mm(dia.). Pertechnetate was eluted from the Dowex-1 column with 5-6 ml of 0.2M Nal followed by washing with 1.5-1.8 ml water. Iodide was removed from this eluate by passage through a 1 g AgCl column (12 mm (H) x 8 mm (dia.)). The final product, pertechnetate, was recovered in 6.5-7.8 ml, while the NaCl content was close to that of normal saline. Each elution operation was complete in approximately 45 min. The mean practical yield was approximately 86% (n = 5). Closed cycle operation with sterile connecting tubes and multi-way stop-cocks was practised. The quality of the [99mTc]NaTcO4 thus obtained complies with the specifications applicable for radiopharmaceutical use. The major merit over other post-elution concentration methods is that the new procedure can function nearly independently of the volume of the primary eluate (in normal saline) from the alumina column generator (ACG). 99mTc generators containing as much as 80-100 g alumina for holding 14.8-18.5GBq (400-500 mCi) 99Mo (at the reference time) are feasible using approximately 15 mg Dowex-1 bed mass. A relatively user-friendly--cum--producer-friendly 99mTc generator system has been demonstrated making use of the inexpensive and easy to prepare (n,gamma)99Mo. The method is also applicable for the more costly 188W-188Re generator.     

Evaluation of stannous oxidation in the preparation of ultrahigh-purity 99m Tc(V)-DMSA

It has been shown previously that renal kit, trivalent technetium-99m dimercaptosuccinic acid [99mTc(III)-DMSA], can be transformed into tumour imaging agent, pentavalent technetium-99m DMSA [99mTc(V)-DMSA], by adding sodium bicarbonate (NaHCO3) and subsequently bubbling with oxygen. However, the purity of this pentavalent preparation was reported to be in the range 83-94% at best. In this study, the preparation of ultrahigh-purity 99mTc(V)-DMSA is described, and the role of stannous oxidation in the conversion of 99mTc(III)-DMSA to 99mTc(V)-DMSA is evaluated in order to understand the underlying mechanism. The results show that controlled oxygen bubbling increases the 99mTc(V)-DMSA levels, with a concomitant decrease in the 99mTc(III)-DMSA levels, in a time-dependent pattern. The purity of the pentavalent DMSA is shown to be consistently very high (>99%), as estimated by chromatography, and this correlates very well with the minimal or no renal uptake of this compound in patient studies.     

Instant 99mTc-labelled glucoheptonate kit for kidney imaging

Gelchromatography column scanning has been used to study the fractions of reduced hydrolyzed 99mTc, 99mTc-pertechnetate and 99mTc-chelate in a 99mTc-glucoheptonate (GH) preparation. A stable high labelling yield of 99mTc-GH complex in the radiopharmaceutical has been obtained with a concentration of 40-50 mg of glucoheptonic acid-calcium salt and not less than 0.45 mg of SnCl2.2 H2O at an optimal pH between 6.5 and 7.0. The stability of the complex has been found significantly affected when sodium hydroxide solution was used for the pH adjustment. However, an alternative procedure for final pH adjustment of the preparation has been investigated providing a stable complex for the usual period of time prior to the injection. The organ distribution and the blood clearance data of 99mTc-GH in rabbits were relatively similar to those reported earlier. The mean concentration of the radiopharmaceutical in both kidneys has been studied in normal subjects for one hour with a scintillation camera and the results were satisfactory.     

Development and biologic evaluation of a kit for preformed chelate technetium-99m radiolabeling of an antibody Fab fragment using a diamide dimercaptide chelating agent

A kit has been developed for 99mTc antibody radiolabeling via defined chemistry using an N2S2 diamide dimercaptide bifunctional chelating agent and the performed chelate method. The process involved efficient transchelation of 99mTc from gluconate to 2,3,5,6-tetrafluorophenyl 4,5-bis-S-(1-ethoxyethyl) mercaptoacetamidopentanoate as an active ester ligand and subsequent conjugation to antibody lysine amine functional groups. The use of the ethoxyethyl group for sulfur protection allowed optimum yields of 99mTc N2S2 chelate formation with complete retention of the active ester. Subsequent addition of antibody Fab fragment gave 99mTc chelate conjugates indistinguishable from the stepwise in situ esterification and purification of the 99mTc N2S2 complex followed by conjugation as previously shown to give stable 99mTc antibody fragments with retained immunoreactivity and tumor-targeting properties.     

The behavior of 99mTc-hexamethylpropyleneamineoxime (99mTc-HMPAO) in blood and brain

99mTc-hexamethylpropyleneamineoxime (99mTc-HMPAO) is a reagent for scanning cerebral blood flow. We investigated how 99mTc-HMPAO changed in the blood and brain. The 99mTc-HMPAO, which was prepared by adding of 99mTcO4- to HMPAO and Sn(II), consisted of primary and secondary complexes, reduced hydrolyzed 99mTc, and 99mTc-pertechnetate. The percentage of the primary complex in 99mTc-HMPAO decreased with time after preparation. The primary complex converted to the secondary one very rapidly in the presence of plasma. When 99mTc-HMPAO was injected into patients, 99mTc activity was immediately partitioned in the plasma fraction, with approximately 60% in whole blood. In plasma, 99mTc was found to be associated with proteins such as albumin and globulin. 99mTc trapped in red cells was not washed out with either plasma or saline. Biodistribution studies showed that the less lipophilic compounds of 99mTc-HMPAO could not pass through the blood brain barrier (BBB), and therefore did not accumulate in the brain. The results of gel chromatography and equilibrium dialysis indicated that no specific 99mTc binding protein was present in the brain. Considering the instability of 99mTc-HMPAO in vivo, we proposed that the speed at which the primary complex converted to the less lipophilic compounds was important in allowing 99mTc-HMPAO to pass through the BBB and to be fixed in the brain.     

Comparison of 99mTc complexes for renal imaging.

The distribution of 17 different agents for renal imaging was compared in the rabbit by organ radioassay at 1 hr. Similarly, 99mTc complexes of iron-ascorbate, glucoheptonate (GHA) and 2,3-dimercaptosuccinic acid (DMS), and 203Hg-chlormerodrin were compared in the dog. The distribution of 99mTc-GHA and DMS was assessed in the human by blood and urinary clearance, external renal measurements, and scintillation camera imaging, and compared with older renal radiopharmaceuticals. Radiation dose estimates, based chiefly on human data, were calculated. Technetium-99m-DMS reaches a high concentration in the renal cortex and its urinary excretion rate and blood clearance are slow. It is excellent for imaging the renal parenchyma without activity in pelvocalyceal collecting system. However, it readily oxidizes and must be used within 30 min of preparation. The biologic distribution of 99mTc-GHA is similar to gluconate and iron-ascorbate complex. Its renal concentration is not as great as that of DMS but its blood and urinary clearances are much faster, resulting in lower radiation doses to most organs. Early camera images with this agent usually demonstrate both the renal parenchyma and collecting system. In later images, ther is excellent demonstration of the parenchyma alone, superior to that obtained with 99mTc-Sn-DTPA. It is a very stable complex and may be used for at least 5 hr after preparation. All radioactive renal agents examined to date have a significant concentration in the liver, making an accurate quantitative comparison between the two kidneys difficult.     

Exploratory study of 99mTc-EC20 imaging for identifying patients with folate receptor-positive solid tumors.

99mTc-EC20 is a folate receptor (FR)-targeted imaging agent consisting of the vitamin folate conjugated to 99mTc. FR is expressed on a variety of epithelial cancers, with advanced cancers often expressing FR at significantly higher levels than earlier stages of the disease. The goals of this pilot study were to determine the percentages of various solid tumors that accumulate 99mTc-EC20 in vivo and to correlate 99mTc-EC20 uptake with immunohistochemistry (IHC) analysis of FR expression in available biopsied tumor tissue. METHODS: A total of 154 patients with proven or suspected cancer and at least one lesion of > or =1.5 cm underwent imaging with 99mTc-EC20. The majority of these patients (77%) had a diagnosis of renal cell carcinoma. The remaining patients had a variety of other solid tumors. Whole-body planar images were obtained 1-2 h after injection, followed by SPECT of the region containing index lesions. The uptake of 99mTc-EC20 in tumors was scored as no uptake, mild uptake, or marked uptake. The resultant 99mTc-EC20 data were analyzed for correlation with the expression of the alpha-isoform of FR, as determined by IHC analysis, in tissue available from prior or subsequent surgery or biopsy. RESULTS: The administration of 99mTc-EC20 was well tolerated. Tumors with increased 99mTc-EC20 uptake were identified in 68% of patients, and IHC results were positive for the expression of the alpha-isoform of FR in 67% of patients. The agreement between methods was 61% overall (kappa = 0.096; 95% confidence interval = -0.085 to 0.277), with 72% agreement of positive results and 38% agreement of negative results. CONCLUSION: In vivo imaging with 99mTc-EC20 identified approximately two thirds of patients as having FR-positive tumors. Agreement between imaging and in vitro IHC was poor but was potentially confounded by a lack of correlation between the time of tissue sampling and the time of 99mTc-EC20 imaging, the heterogeneous expression of FR in metastatic lesions from the same patient, and the inability to detect the beta-isoform of FR by IHC. This pilot study of 99mTc-EC20 scintigraphy indicates that the agent is safe and well tolerated and that this noninvasive procedure may have utility in selecting patients likely to benefit from FR-targeted therapy.     

Preparation and characterization of technetium complexes with Schiff base and phosphine coordination. 1. Complexes of technetium-99g and -99m with substituted acac2en and trialkyl phosphines (where acac2en = N,N'-ethylenebis[acetylacetone iminato])

A series of 23 technetium(III) complexes of the type [TcL(PR3)2]+, where L represents a tetradentate Schiff base ligand in the equatorial plane and PR3 represents the axial phosphine ligands, are reported. Full ligand syntheses and characterizations are included. The technetium complexes were prepared with 99mTc to study the organ distribution in guinea pigs at 5 and 60 min postinjection. Four prototypical complexes of the series were also prepared with either 99gTc or 99gTc/99mTc (designated as carrier-added) to allow macroscopic characterization. Equivalence of the 99gTc and 99mTc complexes was demonstrated by dual detection high performance liquid chromatography (HPLC) techniques. The development of a one-step preparation from the standard two-step method is discussed for some complexes. Biodistribution data are related to structure and lipophilicity. None of the complexes in the series exhibited a tendency for in vivo reduction. Myocardial uptake was favorable for a number of complexes. The optimal agent from this series for further imaging development was chosen based on myocardial uptake, rapid blood and liver clearance, and ability to be formulated as a one-step kit.     

Preparation and evaluation of 99mTc(V)-DMSA complex: studies in medullary carcinoma of thyroid

Consequent to the promising results reported with 99mTc(V)-DMSA for imaging certain types of soft tissue tumors, we have developed methods to prepare this radiopharmaceutical in three ways: from freshly prepared reagents, through the use of a two component kit and use of the standard renal DMSA kit by a modified recipe. The 99mTc(V)-DMSA complex has been subjected to paper electrophoretic and chromatographic procedures and also biodistribution studies. The distinctly different behaviour of this new product compared to that of the well known renal DMSA complex has been clearly established. Scintiimaging in a preliminary clinical trial in patients with medullary carcinoma of the thyroid has been encouraging.     

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.     

Labeling and testing of 99mTc-streptokinase for the diagnosis of deep vein thrombosis.

A detailed study has been made of the method for labeling streptokinase with 99mTc in order to get a radioactive indicator for rapid scintigraphic visualization of thrombi and emboli. The best method found for preparing 99mTc-streptokinase was by reducing 99mTc-pertechnetate with 2 mumole SnCl2 at pH 0.7 and then adding 50,000-75,000 IU of streptokinase at a final pH of about 2. After 1 hr of equilibration the labeling efficiency was 75-80% as determined by the method of gel chromatography column scanning. The dynamic behavior and distribution of 99mTc-streptokinase in different organs was studied in rabbits where a high uptake was found in liver. In a clinical investigation using 99mTc-streptokinase prepared according to this work, however, the liver uptake in man was only 10-20%.     

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.     

Preparation, analysis and application of [99mTc]Human Albumin Microspheres ([99mTc]HAM) for lung scanning

Kits were developed for the sterile labelling of Human Albumin Microspheres with 99mTc. The microspheres were prepared, sieved to get the desired particle size and autoclaved for sterilization. The spheres were treated with stannous chloride and the pH of the suspension was adjusted to 3.7 with phosphate buffer. After freeze-drying the contents of single reaction vials from different batches were reacted with 99mTc and the radiochemical yield (higher than 97%) was determined. The HAM kits were stable and the stability of [99mTc]HAM was followed for 5 h. Lung and liver uptake in mice was determined to be about 90 and 1% respectively. The preparation of [99mTc]HAM is performed in a single step process and excellent human lung scans were obtained.     

Use of technetium-99m(V)thiocyanate to measure gastric emptying of fat

Technetium-99m(V)thiocyanate was evaluated as a radio-pharmaceutical for measuring gastric emptying of fat. Olive oil was labeled with 99mTc(V)thiocyanate by direct extraction from acidic thiocyanate solution. After incubation with dilute HCl (pH 1.4) at 37 degrees C for 3 hr, approximately 5% of the total radioactivity eluted into the aqueous phase. When incubated with human gastric juice (pH 1.8 and 2.2), approximately 8% of the activity was detected in the aqueous phase at 3 hr. Scintigraphic studies performed in two rabbits showed that olive oil labeled with 99mTc(V)thiocyanate emptied slowly from the stomach, with a gastric half-emptying time (T50) of more than 3 hr. A low-nutrient soup labeled with 113mIn-DTPA and mixed with 99mTc(V)thiocyanate labeled oil was consumed by six human volunteers. The oil emptied much more slowly (p less than 0.02) (median T50 = 198 min) than the aqueous component (median T50 = 30 min). These observations indicate that 99mTc(V)thiocyanate is a suitable pharmaceutical to measure gastric emptying of extracellular fat.