Technetium-99m-EC and Other Potential New Agents in Renal Nuclear Medicine

Transcription

1 Technetium-99m-EC and Other Potential New Agents in Renal Nuclear Medicine Justin K. Moran Search for the ideal radiopharmaceutical to measure effective renal plasma flow (ERPF) has been underway since the early 1960s. Although ortho-iodohippuran (OIH) has biological properties suited for measurement of ERPF, the imaging characteristics are less than desirable. With the advent of the molybdenum-99/ technetium-ggm generators, efforts have focused on the development of a technetium-99m agent to measure ERPF. Over the last 10 to 15 years several promising technetium-99m renal imaging agents have been developed. Early examples of technetium-9gm renal agents such as ssmtc-co2dads and 9SmTc-PAHIDA, although not ideal replacements for OIH, demonstrated that a technetium-ggm complex could be actively transported by the renal tubules and provided the impetus for development of new technetium-99m renal agents. The next breakthrough in technetium- 99m renal agents was the development of the triamide mercaptide class of chelating agents by Fritzberg et al. To date the most promising compound V ARIOUS RADIOLOGICAL methods are available to address anatomical questions about the kidneys. However, to determine the differential function of each kidney, and to detect obstruction in urine flow, renography using radiotracers is the method of choice. Measurement of renal plasma flow requires that the agent being used be completely extracted by the kidneys. To date no compound has been found that is completely extracted by the kidneys, and only para-aminohippuric acid (PAH) comes close. Because measurement of effective renal plasma flow (ERPF) using PAH is a time-consuming procedure that requires complex separation methods and cannot be imaged clinically, Tubis et al developed ortho-iodohippuric acid (OIH), a radiolabeled analog of PAH for measurement of ERPF and imaging.l Although OIH yields a good approximation of renal plasma flow, the 364 kev photon of iodine-131 results in poor spatial resolution and the emission of a beta particle increases the radiation burden to the patient. Labeling OIH with iodine-123 results in a better imaging agent, but the availability and cost of iodine-123 makes this labeling problematic. Over the last 20 years there has been considerable work devoted to the development of the "ideal" radiopharmaceutical to measure ERPF and perform renal imaging. Due to the favorable physical properties of technetium-99m (monochromatic energy emission, short in this class is mercaptoacetyltriglycine (MAG3). 99mTC" MAG3 is currently the agent of choice, but it is by no means the perfect replacement for OIH. Problems with high plasma protein binding and clearances that are only 50% to 60% of the OIH clearance make measurement of ERPF difficult. The serendipitous discovery that metabolites of the brain agent sgmtc-ethylenedicysteine diethylester (99mTc-L,L-ECD) are rapidly excreted in the urine has led to the evaluation of sgmtc-l,lethylenedicysteine (sgmtc-l,l-ecd) as a potential renal imaging agent. Studies that have evaluated 99mTC- L,L-EC in animals, normal human volunteers, and patients with various renal disorders reveal that the renal clearance of 99mTc-L,L-EC is higher than ssmtc- MAG3 and more closely approaches that of OIH. Other approaches that are being examined in the development of the ideal renal imaging agent include substitution of various amino acids for glycine residue in MAGa and technetium-99m labeled organic cations. Copyright by W.B. Saunders Company physical half-life, low cost and high availability) most research has been directed towards the development of a technetium-99m-based imaging agent (Fig 1), Generally, technetium forms stable complexes with ligands that contain functional groups such as -COOH, -OH, -NH2, and -SH. Early attempts to create a technetium-99m-based renal imaging agent focused on the diamide dithiolate (DADT) ligand system. 99mTc-N,N'-bis(mercaptoacetyl)-2,3-diaminopropanoate (99mTc-CO2DADS) has favorable renal clearance properties, but labeling required HPLC purification to separate the mixture of stereo-isomers which made routine preparation inconvient. 2 Attempts to develop a technetium-99m labeled analog of PAH by incorporation of an iminodiacetic acid (IDA) moiety yielded p- [(biscarboxymethylaminomethyl)carbamino]hippuric acid (PAHIDA) which had a clearance of less than 50% of OIH. 3 A little over 10 years ago, Fritzberg et al developed the triamide mercaptide (N3S) class of chelating agents to complex technetium. 4 The most successful N3S From the Department of Nuclear Medicine, Albert Einstein College of Medicine and Montefiore Medical Center, Bronx, NY Address reprint requests to Justin K. Moran, PhD, Department of Nuclear Medicine, 1695A Eastchester Road, Bronx, NY Copyright by W.B. Saunders Company /99/ /0 Seminars in Nuclear Medicine, Vol XXlX, No 2 (April), 1999: pp

2 92 JUSTIN K. MORAN A C HO2C -~ B OH H SH HS D O N~ ~ O O O~ N NH H SH HN 0 CO2H Fig 1. Structure of hippuran (A) and selected renal imaging agents CO2DADS (B), PAHIDA (C), and MAG3 (D). ligand to date is mercaptoacetyltriglycine, and 99mTc-mercaptoacetyltriglycine (99mTc-MAG3) is currently the agent of choice for the evaluation of transplant kidney, tubular necrosis, and kidney function in nuclear medicine. However, MAG3 is not an ideal replacement for OIH due to high plasma-protein binding (60% to 80%), and clearance in humans that are only 60% to 65% of OIH, making determination of ERPF complicated. In addition, the preparation of 99mTc-MAG3 requires the kit to be heated at 100~ for 10 minutes which adds an inconvenient step in the preparation. While studying the biodistribution of the metabolites of the brain agent 99mTc-L,L-ethylenedicysteine dietheylester (99mTc-L,L-ECD), Verbruggen et al found that the most polar metabolite was excreted rapidly and efficiently in the urine. This observation had been noted by others, and was assumed to be due to formation of the diacid 99mTc-L,L-ethylenedicysteine (99mTc-L,L-EC) from de-esterification of 99mTC-L,L-ECD.5 CHEMISTRY AND ANIMAL STUDIES L,L-ethylenedicysteine (L,L-EC) was first synthesized by Blondeau et al in 1967, but it was not until the early 1990s that the technetium-99m complex was prepared. 6 99mTc-L,L-EC belongs to the diaminodithiol (DADT) family of chelating agents that contain two amine and two thiol functional groups. DADT compounds have been shown to form very stable complex with the Tc(V)O core through binding of the two thio-sulfur and two aminenitrogen atoms. The majority of DADT ligands can easily bind technetium-99m in high yield at physiological ph. Analysis by Verbruggen et al of reaction conditions that result in the highest radiochemical yield of 99mTc-L,L-EC showed that at ph 7.0 only 50% of the radioactivity was in the desired form. When the ph of the reaction solution is increased to 10-12, the labeling yields increases to 97% to 99%. The cause for low labeling yields at ph less than 10 is due to protonation of the amine nitrogens. At ph less than 10, protons compete with technetium for binding to the nitrogen atoms. As the ph is increased, the amine nitrogens are deprotonated, thus allowing for complexation of technetium-99m. Once the 99mTc-L,L-EC complex is formed at high ph, the ph of the solution can be adjusted to 7.4 without disassociation of technetium- 99m from ethylenedicysteine. After neutralization, 99mTc-L,L-EC can be stored at room temperature for up to 8 hours without any appreciable degradation as determined by HPLC analysis. At high ph (> 12) the complexation is rapid with yields of 99% within 2 minutes. Other factors that have been optimized by Verbruggen et al include the amount of ethylenedicysteine and stannous chloride required (1 mg and 10 lag respectively) for greater than 99% radiochemical yield. 6 Animal studies of 99mTc-L,L-EC have been performed in a variety of species. Initial animal studies in mice comparing 99mTc-L,L-EC, 99mTc-MAG3 and 131I-Hippuran revealed that 99mTc-L,L-EC had a slightly higher urinary excretion at 10 and 30 minutes postinjection compared with 99mTc-MAG3 and that the urinary clearance more closely approaches that of OIH. 6 The liver and intestinal uptake of 99mTc-L,L-EC in mice was significantly lower than that of 99mTc-MAG3 ( % vs % and % vs % injected dose respectively), but was not significantly different than that of OIH. To determine if 99mTc-L,L-EC is primarily excreted by active tubular transport, clearance studies were performed in mice that received 25 mg/kg of probenecid 10 minutes prior to administration of 99mTc-L,L-EC. Probenecid is transported by the renal tubules, and it is used in competitive inhibition assays. In the probenecid treated animals there was a 25% reduction in the urinary excretion of 99mTc-L,L-EC from % to % injected dose. As a consequence of decreased urinary excretion, the hepatobilliary excretion increased (1.86 _ 0.19% vs 4.11 _+ 0.58% injected dose) and the blood clearance decreased (1.97_ 0.24% vs 6.79_ 0.48% injected dose). 6 These results indicate that 99mTc-L,L-EC is probably transported by the same carrier proteins responsible for the excretion of the hippurate anion. The two previous technetium-99m renal tubular

3 9gmTo-EC IN RENAL NUCLEAR MEDICINE 93 agents 99mTc-MAG3 and 99mTc-CO2DADS both contain a carbonylglycine moiety (-CO-NH-CH2- COOH) which is believed to be essential for recognition of these compounds by the receptor proteins of the tubular transport system. 7 It is the carbonylglycine group that mimics the sidechain of hippuran that allows these compounds to be secreted by the tubules. However, 99mTc-L,L-EC does not contain a carbonylglycine moiety, but instead contains two oxotechnetium-glycine sequences (TcO-NH-CH2-COOH) which also appear to mimic the sidechain of hippuran. It was believed that the carbonylglycine group was required for recognition by the transport receptor, but it now has become evident that a free carboxylate is one of the key features needed for receptor recognition. Not only must the complex contain a free carboxylate for recognition by the transporter proteins, but the carboxylate must be on the same side of the molecule (syn) relative to the oxo group of the oxotechnetium core. In 99mTc-MAG3 the terminal carboxylate group can freely rotate which enables the carboxylate to take a syn orientation relative to the oxo group. 99mTc-CO2DADS can exist in conformations in which the carboxylate is in either a syn or anti configuration, but only the syn conformer is recognized by the tubular transporter receptor and is rapidly excreted in the urine. 2 Since 99raTc- L,L-EC was first prepared from the brain agent 99mTc-L,L-ECD, preliminary studies have focused on the L,L-isomer of ethylenedicysteine. However, ethylenedicysteine contains two optically active centers which means that there are four different stereo-isomers possible; L,L,D,D, syn-d,l, and anti-d,l (Fig 2). Both the L,L and D,D-isomers of ethylenedicysteine contain a carboxylate syn to the oxo group. The syn-d,l-isomer of ethylenedicysteine contains two carboxylates syn to the oxo group, Fig 2. \./\,] 99mTc.L,L-EC HN/'---"~N ( \!/'r k./\/ 99rnTc-D,D.EC ~N \=/\=/ 99raTc.syn-D,L-EC NH'~"~N \./ ~ 9~aTe.anti.D,L-EC The four slmctursl isomers of S~mT while the anti-d,l-isomer has no carboxylates syn to the oxo group. Taylor et al reported that when rhenium-d,l-ethylenedicysteine (Re-D,L-EC) was prepared under high ph conditions, only the syn- D,L isomer was formed, s Under neutral conditions, both the syn and anti-re-d,l-ec isomers were isolated and the anti-re-d,l-ec isomer could be converted into the syn-re-d,l-ec isomer at high ph, which did not reform when the ph was lowered. The syn-re-d,l-ec isomer could not be converted into the anti isomer at any ph, which explains why only the syn-d,l-isomer was isolated at high ph. The same phenomenon has been observer with the technetium-99m analogs. 9 Examination of the clearance of 99mTc-L,L-EC, 99mTc-D,D- EC, and syn- 99mTc-D,L-EC in rats revealed that the D,D-isomer had the highest clearance followed by the syn-d,l and the L,L-isomers. Both the L,L and D,D-isomers had similar plasma protein binding (66% and 64%, respectively), while the protein binding for the syn-dl-isomer was 74%. 9 The clearance of the D,D-isomer relative to OIH was 108%, which is significantly higher than the clearance of the L,L and D,L-isomers which were 60% and 67% respectively. The differences in clearance may be due to retention of activity in the kidney. Both the L,L and syn-d,l-isomers had significantly higher retention of activity in the kidneys 22 minutes postinjection (24.4% and 19.3% injected dose, respectively) compared with the D,D-isomer (5.4% injected dose). There has been one report in which the urinary clearance of 99mTc-L,L-EC was less than that of 99mTcMAG3.10 Ozker et al found that in rabbits that the urinary clearance of 99mTc-L,L-EC was 81% of the OIH clearance, while the 99mTc-MAG3 clearance was 93% of the OIH clearance. However, this 99mTc-MAG3 clearance is significantly higher than normally reported, while the 99mTc-L,L-EC clearance was similar to what was reported in other species. The plasma protein binding of 99mTc- L,L-EC was the same as OIH (51%), which was significantly less than that of 99mTc-MAG3 (75%). In baboons 99mTc-L,L-EC and 99mTc-MAG3 had similar times to reach maximum renal activity (Tmax, 2.0 min vs 2.5 min), and similar time from maximum to 50% of peak (Tmax/2, 1.7 min vs 2.2 min). However the plasma protein binding was significantly lower for 99mTc-L,L-EC (28% vs 92%), which resulted in a larger volume of distribution for 99mTc-L,L-EC (1,964 ml vs 789 ml) compared

4 94 JUSTIN K. MORAN with 99mTc-MAG3. The net effect is an increased clearance of 99mTc-L,L-EC compared with 99mTC- MAG3 (541 vs 320 ml/min/1.73 m3). Compared with the gold standard OIH, clearance of 99mTc- L,L-EC was 75.4% of the OIH clearance measured in the same animals, which was significantly more than the 51.7% clearance of 99mTc-MAG3 relative to OIH. 5 STUDIES IN NORMAL VOLUNTEERS Although animals are good models for predicting the behavior of technetium-99m radiopharmaceuticals in vivo, differences between species do occur and the best way to determine the biological properties of new radiopharmaceutical is by using human volunteers. Initial human studies used 99mTc- L,L-EC that was purified by HPLC. This was done not because of impurities in the preparation, but in order to guarantee maximal safety during the first use in humans and is only mentioned for completeness of review. A study of six normal volunteers comparing 99mTc-L,L-EC and 99mTc-MAG3 showed that there were no significant differences in the renograms between the two agents.11 Consequently, the parameters derived from the renograms, including time to maximum renal activity, time from maximum to 50% of peak, and maximal renal uptake as a percentage of the injected dose, were not significantly different. One major difference between 99mTc-L,L-EC and 99mTc-MAG3 was the plasma protein binding. The plasma protein binding of 99rnTc-L,L-EC was significantly lower than 99mTc-MAG3 (31-6.8% vs %), and was about half of OIH (68 _+ 3.4%). A consequence of reduced protein binding is an increase in the volume of distribution. The volume of distribution of 99mTc-L,L-EC was almost twice that of 99mTc- MAG3 (8194 _ ml vs ml). The larger volume of distribution also resulted in a higher clearance of 99mTc-L,L-EC compared to 99mTc-MAG3 ( vs ml/ min/l.73 m2), but was only 75% of the OIH clearance. Plasma clearance of 99mTc-L,L-EC in the first 2 minutes was about 1.5 times higher than the plasma clearance of 99mTc-MAG3 and was similar to the clearance of OIH. From 35 to 60 minutes, the plasma clearance of the two technetium-99m tracers was not significantly different. The high plasma clearance of 99mTc-L,L-EC in the first few minutes can be explained in part by increased glomerular filtration due to lower protein binding, but other factors may also contribute. In Taylor's study comparing the three isomers of ethylenedicysteine in normal volunteers 99mTc-L,L- EC, 99mTc-D,D-EC and syn-99mtc-d,l-ec had renograms that were similar, although 99mTc-D,D-EC had a slightly faster time from peak activity to 50% of peak. 9 There was considerable variation in the plasma protein binding of the three isomers. The D,D-isomer had the lowest protein binding at (28%), while the protein binding of the syn-d,lisomer (72%) was closer to that of 99mTc-MAG3 and OIH. The protein binding of the L,L-isomer (47%) was slightly higher than reported by Verbruggen, but still significantly lower than 99mTc-MAG3 and OIH. 11 As seen in the previous study, low protein binding results in higher clearance rates. The clearance of the D,D-isomer was the highest, and was 82% of the OIH clearance. The L,L-isomer had the second highest clearance which was 70% of the OIH clearance, and was in good agreement with the value reported in the previous study. 99mTc-syn- D,L-EC which had the highest protein binding, had the lowest clearance at 40% of OIH which was less than that reported for 99mTc-MAG3. In 6 normal volunteers, there was no difference in renograms obtained with 99mTc-L,L-EC, 99mTC- MAG3 and OIH. As a result, time to peak activity, and time from peak to 50% of peak activity showed no significant difference between the two technetium-99m agents (Tmax; minutes vs minutes, Tmax/2; minutes vs minutes). The Tma x for OIH was not significantly different than the two technetium-99m agents, but the Tmax/2 was significantly shorter.12 Kibar et al evaluated 99mTc-L,L-EC and 99mTc- MAG3 in normal children and found that there was no difference in the renograms or the parameters derived from the renograms. However, the values for time to maximum activity and time from maximum to 50% ( minutes and minutes, respectively) are slightly shorter than those reported in adults. 13 In a study performed by Gupta et al in normal volunteers, the clearance of 99mTc-L,L-EC ranged from 69 to 85% relative to OIH with a mean of 75%. 14 The time to peak activity and time from peak to 50% of peak were in good agreement with other reported studies. There were no reported adverse reactions in any of the normal volunteers in any of the trials after the intravenous administration of 99mTc-L,L-EC.

5 ~ IN RENAL NUCLEAR MEDICINE 95 STUDIES IN PATIENTS WITH RENAL DISORDERS In normal volunteers the scintigraphic images obtained with 99mTc-L,L-EC were of equal or greater quality as the current agent of choice 99mTc-MAG3. The clearance of 99mTc-L,L-EC is closer to that of OIH than is 99mTc-MAG3, and yields a better approximation of ERPF. However, the true test of a new radiopharmaceutical is how it performs under "battle conditions." The decrease in renal function of patients with various renal disorders can cause marked changes in the distribution of renal imaging agents. To date a number of studies have been performed in patients with a variety of renal disorders. In one of the early studies, Ozker et al compared 99mTc-L,L-EC, 99mTc-MAG3, and OIH in 16 patients with obstructive renal disease who had proven or suspected hydroureteronephrosis. Prior to the study, all patients had undergone uropraphic examinations which demonstrated hydroureteronephrosis and/or urolithiasis. 12 Serum creatinine levels were in the normal range for all patients. Both technetium-99m agents were prepared from kit formulations, and had labeling efficiencies were greater than 94% without any further purification necessary. Iodine-131-OIH was obtained from a commercial source. There was no significant difference observed between the renograms obtained with the three agents, except for slightly higher accumulation of 99mTc MAG3 activity in the liver (Fig 3). Consequently, the parameters derived from the renograms including the time to peak activity (Tmax) and the time from peak to 50% activity (Tmard2) were similar. As expected in patients with obstructive renal disease the Tmax (99mTc-L,L-EC; minutes vs min, 99mTc MAG3; minutes vs 4.00 _ minutes, and OIH; minutes vs 4.25 _ 0.37 min), and Tmax/2 (99mTc-L,L-EC; minutes vs min, 99mTc-MAG3; minutes vs minutes, and OIH; 7.58 _ minutes vs minutes) increased for all three agents compared to normal controls. Gupta et al evaluated 99rnTc-L,L-EC in 16 patients with various renal disorders. 14 Of the 16 patients, 10 had suspected obstructive nephropathy, 4 had suspected renal vascular hypertension, and 2 were in acute renal failure. In the 99mTc-L,L-EC studies, the results were found to be concordant with the clinical findings. In 5 patients with chronic renal failure both a 99rnTc-L,L-EC and a 99mTc MAG3 study were performed. There was no significant difference in the image quality or parameters derived from the renograms between the two tracers. Both tracers showed faint hepatic activity at 40 to 50 minutes postinjection, but the extrarenal activity did not interfere with the images during the renogram phase. This study also included the first patients with renovascular hypertension to be studied with 99=Tc-L,L-EC. Even with severely impaired renal function, 99mTc-L,L-EC provided interpretable renographic images that were in concordance with the clinical findings. All four patients with renovascular hypertension were started Fig 3. Corresponding images of Sg~Tc-EC (A) and sgmtc-mag3 (B) in a patient. The delineation of the kidneys is better in the images of 99mTc-EC than those of SSmTc-MAG3 because of lower background and hepatic activity. (Reprinted with permission from the Society of Nuclear Medicine. TM)

6 96 JUSTIN K. MORAN on ACE inhibitors, and their hypertension was controlled without any deterioration in renal function. The two patients with acute renal failure secondary to abdominal aortic aneurysm surgery studied with 99mTc-L,L-EC showed relative preservation of flow and progressive cortical accumulation of tracer in the kidneys. Biopsy confirmed that the cause of the renal insufficiency was acute tubular necrosis, and the patients recovered on conservative management. Kibar et all3 evaluated the utility of 99mTc- L,L-EC in 27 children with various renal disorders including: urinary tract infection, pelviureteric junction stenosis, vesicoureteric reflux, renal agenesis, nephrolithiasis, and neurogenic bladder. The children were divided into two groups. In group 1, imaging was performed with both 99mTc-L,L-EC and 99mTc MAG3; in group 2, only 99mTc-L,L-EC imaging was performed. In group 1, in which both 99mTc-L,L-EC and 99mTc MAG3 scintigraphy was performed, there was no significant difference in the Tma x or Tmax/2 ( minutes vs minutes, and minutes vs 10.8 _ 4.3 minutes, respectively) between the two technetium agents. However, the Tmax for four children was not reach with 99mTc-MAG3, but was with 99mTc- L,L-EC during the period of the study. Images obtained with the two agents were similar, except delineation of the kidneys was better with 99mTc- L,L-EC due to lower background activity. In group 2, Tmax ( minutes) and Tmax/2 ( minutes) were slightly lower than the values obtained with 99mTc-L,L-EC in the children in group 1. The investigators believe that in chronic renal failure, even with low glomerular filtration rates, 99mTc-L,L-EC provides better quality images than 99mTc-MAG3 and that 99mTc-L,L-EC should be the agent of choice. An important use of radionuclide scans of the kidney is in the routine evaluation of the renal graft function of patients who have recently undergone kidney transplant surgery. Stoffel et al evaluated the safety and pharmacokinetics of 99mTc-L,L-EC in a large population of renal transplant recipients, and compared the imaging properties with those of 99mTc-MAG3.I5 In their study, the authors divided the patients into two groups. A dual tracer study with 99mTc-L,L-EC and OIH was performed with group 1 which consisted of 25 patients with unstable renal function (recently transplanted [less than 15 days] or patients with posttransplantation complications). Group 2 consisting of 25 patients with stable renal function (long-term follow-up after transplantation) were studied with 99mTc-L,L- EC, 99mTc MAG3, and OIH. To cover a wide range of renal function values, four patients on chronic hemodialysis (3 anuric), one potential living kidney donor and five healthy volunteers were also evaluated using the same procedure as group 1. The radiochemical yields of 99mTc-L,L-EC from kit formulations ranged from 97% to 99.5% and were comparable those obtained for 99mTc MAG3. Overall there was a close correlation between the clearance values of 99mTc-L,L-EC and OIH, with the mean clearance of 99mTc-L,L-EC about 71% of the OIH clearance. In the three anuric patients who were undergoing chronic hemodialysis the clearance of 99mTc-L,L-EC was significantly lower than the clearance of the other patients (43.1% 7.4%). There was no significant difference in the total volume of distribution between the two tracers 99mTc-L,L-EC and OIH. In group 2, the plasma clearance of 99mTC MAG3 was systematically less than 99mTc-L,L-EC, and was approximately 52% of the OIH clearance. The volume of distribution of 99mTc MAG3 was significantly less than both 99mTc- L,L-EC and OIH which can be attributed to the higher plasma protein binding of 99mTc-MAG3 compared to the other two agents. Comparison of the scintigraphic images obtained with 99mTc- L,L-EC and 99roTe MAG3 showed no significant differences, except in the patients with severe renal failure. In the three anuric patients, there was no significant activity in the bowel and gallbladder, and minimal activity in the liver in the 99mTc- L,L-EC studies, whereas there was significantly increased liver activity in the 99mTc MAG3 studies. There were no differences in Tma x and Tmard 2 between 99mTc-L,L-EC and 99mTc MAG3 in the group 2 patients. Technetium-99m-L,L-EC captopril scintigraphy has been used by several groups for the diagnosis of renovascular hypertension. 16,17 Over the last 2 years Ugur et al have used 99mTc-L,L-EC captopril scintigraphy in 72 patients with angiographic correlation. 18 The investigators find that the diagnostic criteria of 99mTc-L,L-EC captopril scintigraphy are similar to those of other tubular agents. Namely the worsening in renographic grade or retention of parenchyma activity after captopril intervention compared to baseline suggests high probability for renal artery stenosis. Although there were no

7 9a"Tc-EC IN RENAL NUCLEAR MEDICINE 97 significant differences in the perfusion index, split renal function and effective renal plasma flow pre and postcaptopril, there were significant changes in the Tmax, Tm~2 and residual cortical activity between the pre- and postcaptopril studies. The sensitivity and specificity of 99mTc-L,L-EC captopril scintigraphy in this study to detect renal artery stenosis were reported to be 95% and 98%, respectively. 19 Other Renal Imaging Agents Two of the main drawbacks to 99mTc-MAG3 are that its clearance is only 50% to 60% of OIH, and the high plasma protein binding (75% to 90%), thus making measurement of ERPF difficult. MAG3 is a peptide composed of three glycine residues, incorporation of different functional groups into the peptide can be accomplished by substitution of the glycine residues with one of the other 19 naturally occurring amino acids. Substitution of glycine residues with different amino acids makes it possible to alter the charge, and lipophilicity of the resulting technetium-99m complex. Introduction of amino acids which contain a carboxylate functional group on the alpha-carbon such as mercaptoacetylglycylglycylaspartate (MAGGD) and mercaptoglycylglycylglutamate (MAGGE) results in dianionic technetium-99m complexes that are rapidly excreted in the urine. 2~ Interestingly, incorporation of another anionic functional group that results in a trianionic technetium-99m complex results in slower renal excretion. 22 The incorporation of aliphatic or aromatic residues such as alanine or phenylalanine results in complexes that have higher hepatobiliary excretion and decreased rates of renal excretion due to increased lipophilicity. Although there are 8,000 possible ways to combine the 20 naturally occurring amino acids in a tripeptide, incorporation of more than one asymmetric center results in the formation of diasteromers which usually require HPLC purification for separation, and thus limit the number of useful derivatives. Because of the low chemical stability of the thiol group to oxidation, MAG3 is usually synthesized as the S-benzyl protected derivative. It is the protected form of MAG3 that is supplied in commercial kits, and after reformulation the kit must be kept in the dark to prevent oxidation of the thiol group. To overcome this problem Verbruggen et al examined the substitution of a hydroxy group for the thiol group in MAG3, hydroxyacetyltriglycine (HAG3). 23 Initial attempts to label the O-benzoyl protected HAG3 with 99roT at elevated temperatures and ph less than 10 were unsuccessful. An 83% yield was possible when the ph of the solution was adjusted 12 or greater and heated for 10 minutes at 100~ In contrast, starting with the unprotected HAG3, direct labeling at alkaline ph (ph -- 12) and room temperature resulted in yields >95%. Once formed at high ph, the solution could be neutralized without disassociation of technetium-99m from hydroxyacetytriglycine. Because of the increased stability of the hydroxy group compared to the thiol group, it is not necessary to supply HAG3 as the O-benzyl protected derivative. Initial animal studies in mice showed that 99mTc-HAG3 had a slightly higher urinary excretion, a faster renal transit, and a significantly lower hepatobiliary handling than 99mTc-MAG3. In baboons, the clearance of 99mTc- HAG3 was about 62% of the OIH clearance which was significantly higher than clearance of 99mTc- MAG3 (54%). The plasma protein binding of 99mTc-HAG3 was 65%, which was similar to OIH, and less than 99mTc MAG3. Evaluation of 99mTc- HAG3 in a normal volunteer gave similar results as in the baboon with plasma protein binding of 61% and a clearance of 71.5% of the OIH clearance. The more favorable renal clearance of 99mTc-HAG3 can be attributed to its lower plasma protein binding, comparable to what was seen with 99mTc-L,L-EC. Although the renal excretion characteristics of 99mTc HAG3 are slightly better than 99mTc-MAG3 and the labeling can be done at room temperature, the chemical stability of the 99mTc-HAG3 to transchelation is less than the thiol containing analog. Additional work needs to be done on this new class of ON 3 ligands before it is ready for possible clinical applications. Because cystine contains functional groups found in many of the previous renal imaging agents, it was evaluated as a possible replacement for OIH. Technetium-99m forms a stable complex with cystine at room temperature and moderately alkaline ph (ph 8 to 9). The clearance of 99mTc-cystine in rabbits ranged from 75% to 86% of the clearance of OIH. 24 However, in dogs the clearance of 99mTc-cystine was only 47% of the OIH clearance. 25 The plasma protein binding of 99mTc-cystine was 52% + 5%, which was significantly higher than the protein binding of OIH (35% 4%). The higher protein binding of 99mTc-cystine resulted in a smaller volume of distribution compared with OIH

8 98 JUSTIN K. MORAN ( vs ). The low clearance cannot only be blamed on the lower volume of distribution, because 99mTc-MAG3 which has a higher plasma protein binding and lower volume of distribution relative to OIH has a higher clearance than 99mTccystine. It does appear that 99mTc-cystine is handled by the same transporter proteins as OIH because the clearance of 99mTc-cystine can be inhibited by the administration of probenecid. Therefore, some other factors must be responsible for the low clearance of 99mTc-cystine. Misra et al measured the clearance of 99mTc-cystine in both normal volunteers and patients (both transplant and chronic renal failure) relative to OIH and 99mTc-DTPA.26 In all three groups 99mTc-cysfine had clearances 2.0 to 2.5 times higher than 99mTc-DTPA, indicating a significant tubular secretion component. However, the clearance of 99mTc-cystine was only 39% to 42% of the OIH clearance. Although 99mTc-cystine is not likely to replace either 99mTc-L,L-EC or 99mTC- MAG3 as agent of choice to measure ERPF, it does have the unique advantage of being able to determine both function and structure at the same time and may be used as a replacement for 99mTc-DMSA due to retention of activity in the kidney. Gianolli et al27 investigated a compound similar to cystine, the bidentate chelate N-(mercaptoacetyl- )glycine (GAM). GAM contains both a thiolato sulfur and an amido nitrogen similar to MAG3 and DADS, but is a bidentate ligand like DMSA. The resulting technetium-99m complex contains two GAM ligands per technetium-99m (99mTc-2GAM) which can adopt either a cis- or trans-configuration relative to the oxotechnetium core. Biodistribution studies in animals and normal volunteers indicate that 99mTc-2GAM has biological properties more similar to 99mTc-DMSA than to either 99mTc-MAG3 or 99mTc-DADS. 99mTc-2GAM activity in the kidney reaches a plateau more rapidly than 99mTc- DMSA, and thus may be a possible replacement for 99mTc-DMSA. So far, all of the imaging agents discussed are organic anions and thus are transported by the organic anion receptors in the renal tubular system. Nonetheless, problems can arise when uremia develops and there is a buildup of organic anions in the plasma. The accumulation of organic anions can competitively inhibit renal tubular transport of tracers such as OIH, 99mTc-MAG3, and 99mTc-L,L- EC, leading to artificially low estimates of ERPF and tubular function in uremic patients. 28 There is another tubular transport mechanism that has remained largely unexplored due to lack of specific radiopharmaceuticals that is not inhibited by anion accumulation, the cationic transporter system. Herzog et al examined the feasibility of estimating renal function through the use of three novel technetium-99m labeled organic cations in animals (99mTc-fyclam, 99mTc-TAU and 99mTc-TMC).29 The three chelating agents used all belong to the tetraazapolyamine family (Fig 4), and form stable complexes with dioxotechnefium core. The overall charge of each technefium-99m complex was + 1. Radiolabeling yields of >98% were obtained with each chelating agent without further purification necessary. The plasma protein binding for the three _A _+ B _+_C NH NH H3C~ ~/CH3 ( %T~i)/N'~ L.~ N/IOI~N/~J H3C ~ ~ ~CH3 D -12 O H2 Fig 4. Proposed structure of four cationic technetium-99m complexes developed to measure effective renal plasma flow Sg"Tc-Cyclam (A), "mtc-tau (B), 99mTc-TMC (C), and 99mTc-DACH (D).

9 aarntc-ec IN RENAL NUCLEAR MEDICINE 99 organic cations ranged from 32.6% to 38.9%, which is comparable to 99mTc-L,L-EC and significantly less than OIH and 99mTc-MAG3. The renal clearance of the three agents ranged from 12.0 to 16.5 ml/min/kg BW, and were 2.0 to 3.0 times higher than the renal clearance of inulin (5.2 ml/min/kg BW) measured in the same animals. The clearances were approximately 60% the clearance of PAH (24.9 ml/min/kg BW). Because the clearance of OIH is about 85% of that of PAH, the labeled cations clearance would be approximately 70% of OIH, which is similar to 99mTc-L,L-EC. The Tmax and Tmax/2 for 99mTc-cyclam was measured in normal rats and renally impaired rats (impairment was induced by a single 5 mg/kg intraperitoneal injection of the nephrotoxin cisplatin) and compared with 99mTc-MAG3. The Tmax and Tmax/2 for 99rnTc-cyclam ( , and ) were not significantly different than 99raTc-MAG3 ( , and ) in the normal rat. However, in the renally impaired animals, the Tmax for 99mTc-cyclam was higher than 99mTc-MAG3 ( vs min), indicating that 99mTc-Cyclam is handled by the tubule cationic transporter system. Another cationic technetium-99m complex that has been examined as a potential tubular function agent is technetium-99m-l,2-diaminocyclohexane (99mTc-DACH).3~ Initial studies in mice by Solanki et al reveled that 99mTc-DACH had a clearance of 82% of OIH in the same animals, and a low plasma protein binding of _ 0.55%. The clearance of 99mTc-DACH was over five times higher than the 99mTc-DTPA clearance in this study indicating substantial tubular secretion in addition to any glomerular filtration. In the same study, the clearance of 99mTc-DACH was compared to that of 99mTc-MAG3 in three normal human volunteers. The clearance of 99mTc-DACH was slightly less than that of 99raTc-MAG3 ( ml/min vs ml/min). However, the plasma protein binding and the volume of distribution were significantly higher for 99mTc-DACH. Padhy et al determined the clearance of 99mTC- DACH in seven normal volunteers before and after administration of thiamine, a competitive cationic transport inhibitor. 31 The clearance of 99mTc-DACH was only 28% of the OIH, significantly less than was seen in the previous study. The clearance of 99mTc-DACH could be decreased by the administration of thiamine, although the change in 99mTc- DACH was relatively small. Further experiments must be performed to clearly identify the mode of excretion of these tracers. Over the last 10 years the use of receptor based imaging agents in nuclear medicine has increased dramatically. These radioligands can be divided into two main categories; peptide/protein and nonpeptide based ligands. In renal nuclear medicine the emphasis has been on the peptide/protein-based imaging agents. A peptide that has received some attention is atrial natriuetic peptide (ANP), which is a peptide hormone produced in the cardiac atrium that acts upon the kidney playing an important role in fluid, electrolyte, and blood pressure homeostasis. 35 Hamet et al have labeled ANP with iodine- 123 and studied the distribution in both rats and monkeys. 36 The kidneys and lungs are the two main organs expressing receptors for ANP, and accounted for 35% of the total uptake. Uptake could be inhibited by competition with cold ANP and C-ANP indicating that the uptake is receptor mediated. On scintigraphic imaging, the kidneys are clearly visualized by 5 minutes and remain visualized for up to 2 hours. Because of its small size, ANP is rapidly cleared from the blood allowing for early imaging times. Radiolabeled ANP has potential applications in the diagnosis of diseases such as diabetic nephropathy and renovascular stenosis. Radiolabeled monoclonal antibodies have been used in renal nuclear medicine for the detection of infarction and the monitoring of rejection after transplantation. Loutfi et al investigated the use of four different indium-111 labeled antibodies (MRC OX-19, MRC OX-39, MN and F ) for the detection and monitoring of rejection after kidney transplantation in rats. 37 Of the four antibodies investigated, only MN which is directed against the DA class I MHC molecules showed increased uptake in the rejected graft relative to the normal kidney. The ratio of activity in the transplanted kidney relative to the native kidney ranged from to The uptake of MN in the transplanted kidney ranged from 20% to 45% of the injected dose. Further studies need to be performed, but MN is a promising tracer for the evaluation of transplant rejection. Chen et al have investigated the use of radiolabeled TNT-1 an F(ab')2 monoclonal antibody fragment directed against nuclear histone antigens for the detection if kidney infarction. 3s TNT-1 binds to

10 100 JUSTIN K. MORAN necrotic tissue and shows maximal binding in areas that appear viable but that border on areas of necrosis, and presumably contain large numbers of cells in the early phases of cell degeneration. The activity in the infarcted kidney was almost 60 times higher than in the normal kidney. Because uptake appears optimal within hours of injury, this tracer might be useful in detecting various forms of necrosis in focal renal infarction and in acute organ graft rejection. REFERENCES 1. TuNs M, Poshick E, Mordyke RA: Preparation and use of Ia3qabelled sodium iodohippurate in kidney function test. Proc Soc Exp Biol Med 63: , Fritzberg AR, Kuni CC, Klingensmith WC III, et al: Synthesis and biological evaluation of Tc-99m-N,N-bis(mercaptoacetyl)-2,3-diaminopropanoate: A potential replacement for (I- 131)o-iodohippurate. J Nucl Med 23: , Chervu LR, Sandro BM, Blaufox MD: Technetium-99m labeled p-aminohippuic acid analog: A new renal agent. J Nucl Med 25: , Fritzberg AR, Kasina S, Eshima D, et al: Synthesis and biological evaluation of Tc-99m-MAG3 as a hippuran replacement. J Nucl Med 27: , Verbruggen A, Bormans G, Van Nerom C, et al: Isolation of the mono-ester mono-acid derivatives of 99mTc-ECD and their metabolites in mice. In: Nicolini M, Bandoli G, Mazziu (eds): Technetium and Rhenium in Chemistry and Nuclear Medicine 3. Verona: Cortica International and New York, NY: Raven Press, pp , Verbrnggen A, Nosco DL, Van Nerom CG, et al: Technetium-99m-L,L-ethylenedicysteine: a renal imaging agent. I labeling and evaluation in animals. J Nucl Med 33: , Despopoulos A: A definition of substrate specificity in renal transport of organic anions. J Theor Biol 8: , Hansen L, Lipowska M, Taylor A Jr, et al: A new and unexpected arrangement for a rhenium(v)=o(n2s2) complex. The donor set in the basal plane is NOS2. Inorg Chem 34: , Taylor A Jr, Hansen L, Eshima D, et al: Comparison of technetium-99m-ll-ec isomers in rats and humans. J Nucl Med 38: , Ozker K, Kabasakal L, Liu Y, et al: Evaluation of 99Tcm-biscisate as a renal imaging agent. Nucl Med Commun 18: , Van Nerom CG, Bormans GM, DeRoo MJ, et al: First experience in healthy volunteers with technetium-99m-l,lethylenedicysteine a new renal imaging agent. Eur J Nucl Med 20: , Ozker D, Onsel C, Kabasakal L, et al: Technetium-99m- N,N-ethylenedicysteine a comparative study of renal scintigraphy with technetium-99m-mag3 and Iodine-131-OIH in patients with obstructive renal disease. J Nucl Med 35: , Kibar M, Noyan A, Aharat A: 99Tcm-N,N-ethylenedicysteine scintigraphy in children with various renal disorders: A comparative study with 99Tcm-MAG3. Nucl Med Commun 18:44-52, Gupta NK, Bomanji JB, Waddington W, et al: Technetium- 99m-L,L-ethylenedicysteine scintigraphy in patients with renal disease. Eur J Nucl Med 22: , Stoffel M, Jamar F, Van Nerom CG, et al: Evaluation of technetium-99m-l,l-ec in renal transplant recipients: Acom- parative study with technetium-99m-mag3 and iodine-125- OIH. J Nucl Med 35: , Kostadinova I, Simeonova A: The use of 99mTc-EC captopril test in patients with hypertension. Nucl Med Commun 16: , Ugur O, Caner B, Cekirge S, et al: The diagnosis of renovascular hypertension with 99mTc-ethylenedicysteine captopill scintigraphy. Invest Radiol 31: , Ugur O, Peksoy I, Caner B: Technetium-99m-ethylenedicysteine: An alternative agent to detect renovascular hypertension. J Nucl Med 38: , Ugur O, Peksoy I, Caner B, et al: Results of a 2 year prospective study to assess efficacy and methodology of technetium-99m-ec captopril scintigraphy [abstract]. Eur J Nucl Med 24:940, Eshima D, Taylor A Jr, Fritzberg AR, et al: Animal evaluation of Tc-99m triamide mercaptide complexes as potential renal imaging agents. J Nucl Med 28: , Eshima D, Fritzberg AR, Taylor A Jr, et al: Biological studies on a new class of Tc-99m renal tubular function agents. In Billingburst MW (ed): Current Applications in Radiophannacology, Proceeding of the Fourth International Symposium on Radiopharmacology. Elmsford, NY: Pergamam , Eshima D, Fritzberg AR, Taylor A Jr: 99mTC renal tubular function agents: Current status. Semin Nucl Med 20:28-40, Vanbilloen HE Dezutter NA, Cleynhens B J, et al: Characteristics and biological behavior of 99mTc-labelled hydroxyacetyltriglycine, a potential alternative to 99mTc-MAG3. Eur J Nucl Med 24: , Johannsen B, Syhre R, Spies H, et al: Chemical and biological characterisation of different Tc-complexes of cysteine and cystine derivatives. J Nucl Med 19: , Misra M, Sarkar HS, Chakravarty M, et al: 99Tcm-cystine, a renal function and imaging agent: A comparative study in dogs with 13q-hippurate and 99Tcm-glucoheptonate to evaluate its functional and imaging characteristics. Nucl Med Commun 15: , Misra M, Das BK, Gambhir S, et al: Clinical evaluation of Tc-99m cystine a new renal radiopharmaceutical. Clin Nucl Med 19: , Gianolli L, Dosio F, Matarrese M, et al: 99mTc-2GAM: A tracer for renal imaging. Nucl Med Bio123: , Preuss HG, Massry SG, Maher JF, et al: Effects of uremic sera on renal tubular p-aminohippurate transport. Nephron 3: , Herzog KM, Deutsch E, Deutsch K, et al: Synthesis and renal excretion of technetium-99m-labeled organic cations. J Nucl Med 33: , Solanki KK, Britton KE, Bomanji J, et al: Tc-99m labelled cationic complexes---a new class of renal imaging agents--results of preliminary human studies [abstract]. J Nucl Med 32:1105, 1991

11 SgmTc-EC IN RENAL NUCLEAR MEDICINE Padhy AK, Solanki KK, Bomanji J, et al: Clinical evaluation of 99mTc-diaminocyclohexane a renal tubular agent with cationic transport: Results in healthy human volunteers. Nephron 65: , Kabasakal L, Turogla HT, Onsel C, et al: Clinical comparison of technetium-99m-ec, technetium-99m-mag3 and iodine-131-oih in renal disorders. J Nucl MOd 34: , Prvulovich EM, Bomanji JB, Waddington WA, et ah Clinical evaluation of technetium-99m-l,l-ethylenedicysteine in patients with chronic renal failure. J Nucl Med 28: , Kabasakal L, Atay S, Vural VA, et al: Evaluation of technetium-99m-ethylenedicysteine in renal disorders and deterruination of cxtraodon ratio. J Nucl Med 36: , Atlas SA, Laragh JH: Atrial natriuretic peptide: a new factor in hormonal control of blood pressure and electrolyte homeostasis. Ann Rev Med 37: , Lambert R, Willenbrock R, Tremblay J, et al: Receptor imaging with Atrial natriuretic peptide part 1: high specific activity iodine-123-atrial natriuretic peptide. J Nucl Med 35: , Loutfi I, Batchelor JR, Lavender JP: Imaging and Quantiration of renal transplant rejection in the rat by in vivo use of rain labelled antilymphocyte and anti-class I and II major histocompatibility complex monoclonal antibodies. Tranplantation Proc 23: , Chen F, Wisner JR, Omachi H, et al: Localization of monoclonal antibody TNT-1 in experimental kidney inforction of the mouse. FASEB 4: , 1990