SHORT COMMUNICATION 
Annals of Nuclear Medicine Vol.13, No.2. 127-132. 1999 
Technetium-99m complex of N-(2-pyridylmethyl)iminodiacetic acid as a new renal radiopharmaceutical 
Yoshiharu KARUBE, Koji IWAMOTO and Jjro TAKATA 
Faculty of Pharmaceutical Sciences, Fukuoka University 
A tetradentate chelating agent constituting of an iminodiacetic acid group and a nitrogen atom of pyridine, N-(2-pyridylmethyl)iminodiacetic acid (PMIDA), was coordinated with 99mTc and evaluated as a renal functional agent. The complex of PMIDA with 99mTc was prepared by using a stannous chloride solution as a reducing agent. The chelating efficiency was analyzed by thin layer chromatography and electrophoresis. Chelation with 99mTc resulted in a single radiochemical product. Biological studies were performed in mice and rats 99mTc-PMIDA was removed from the circulation solely by the kidneys. Clearance of 99mTC-PMIDA from the blood and the kidneys was as rapid as that of 99mTc-diethylenetriaminepentaacetic acid. The rate of blood clearance was unaffected by the administration of probenecid (a test for tubular secretion by the weak-acid mechanism), so that the glomerular filtration rate could be estimated by measuring its clearance from the blood. The results in animals with myohemoglobinuric acute renal failure suggested that 99mTc-PMIDA might be a useful renal function radiopharmaceutical. 
Key words: 99mTc; N-(2-pyridylmethyl)iminodiacetic acid, renal agent, radiopharmaceutical 
INTRODUCTION 
A NVMBER of radioactive glomerular filtration rate (GFR) agents have been developed and a variety of satisfactory agents are now available Including 99mTc-diethylenetri-aminepentaacetic acid (DTPA),1 169Yb-DTPA,2 51Cr-ethylenediaminetetraacetic acid3 and 125I-iothalamate,4 Among those widely used clinically is 99mTc-DTPA because of the nuclide 99nTc has such ideal physical properties for imaging and gives a low patient radiation dose per imaginable photon. 
DTPA is very stable and also very polar due to the presence of five carboxylic groups and three amino groups. 99mTc-DTPA routinely obtained by complexation with stannous ion appears to be a single radiochemically pure species, since a variety of radioanalytic separations all yield a single fraction.5 But values reported for its stability 
Received September 9, 1998, revision accepted January 18, 1999. 
For reprint contact: Yoshiharu Karube, Ph.D., Faculty of Pharmaceutical Sciences. Fukuoka University, Nanakuma 8-19-1, Jonan-ku, Fukuoka 814-0180, JAPAN. 
E-mail: karube@fukuoka-u.ac.jp 
constant range from 1017 to l026,6-8 which lets us suppose that complexes with different structures were involved in the respective preparations 99mTc-DTPA has a double negative charge depending on uncoordinated carboxylic groups.9 The uncoordinated carboxylic groups and amino groups bring the possibility of complicated chelating reactions and ionic plasma protein-binding reaction.9-13 The chelation of cytosolic ionized calcium ion may produce cell toxicity and ion deregulation in the kidney and bronchus.14 With a view to finding better renal functional agents with low protein binding and low toxicity, we synthesized many iminodiacetate ligands for 99mTc and evaluated them as potential renal functional agents. 
Davison and coworkers developed a new class of tetradentate N2S2 ligands for 1 : 1 chelation with pentavalent Tc.15 The highly stable chelate of N,N'-bis(s-benzoylmercaptoacetyl)ethylenediamine with a [Tc = 0] core became known as the Tc-DADS complex 16 In view of this, it is suggested that a tetradentate chelating agent constituted on iminodiacetic acid and nitrogen atom of pyridine, N-(2-pyridylmethyl)iminodiacetic acid (PMIDA), also may form a highly stable chelate with 99mTc. Although the stability constant (4.92) of calcium-PMIDA was lower than that (10.74) of calcium-DTPA, PMIDA was easily complexed with 99mTc by using stannous ion as the reductant. The present paper is concerned with the in vivo behavior of this 99mTc complex and its relation to chemical structure. The findings show that the PMIDA complex offers a promising new agent as a renal radiopharmaceutical. 
MATERIALS AND METHODS 
Materials 
Technetium-99m pertechnetate (99mTcO4-) was eluted from a sterile 99Mo-99mTc shielded generator (Daiichi Radioisotope Laboratories) with isotonic saline. ortho-131I-iodohippuran (131I-OIH) was obtained from Daiichi Radioisotope Laboratories. PMIDA was synthesized by the method described previously.17 PMIDA recrystallized from aqueous ethanol, had m,p. 227-230deg.C (Anal. Calcd for C10H12N2O4 M.224.2: C, 53.57; H, 5.39; N, 12.49%. Found: C, 53.31; H, 5.36; N, 12.32). N-(Benzyl)-iminodiacetic acid, N-(3-pyridylmethyl)iminodiacetic acid and N-(4-pyridylmethyl)iminodiacetic acid were synthesized by a similar method. Other materials were purchased from commercial sources. 
Formation of 99mTc complex 
A solution containing 0.05 M of PMIDA in 1 ml was adjusted to pH 2.0-11.O with 0.1 M NaOH or 0.1 M HCl. A solution (pH 7,4) containing 0.1 M-0.005 M of PMIDA in 1 ml was prepared. A solution (pH 7.4) containing 0.02 M DTPA was also prepared. A 0.1 ml of freshly prepared solution (2.0 mg/ml in 0.1 M HCl) of stannous chloride (NAKARAI Chemicals Ltd.) was added to the ligand solution and the pH was readjusted to the pH for the purpose (pH 2.0-11.0 or 7.4). The resulting solution was passed through a 0.45 um membrane filter (Millipore) into a sealed vial. Pertechnetate (99mTcO4-) (55.5 MBq, 1.0-2.0 ml) was added. The mixture was shaken gently and allowed to stand for 10 min at room temperature. 
Chemical studies and Physical characteristics of 99mTc-PMIDA 
The efficiency of complexation with 99mTc was evaluated chromatographically with a 0.25 mm Silica-gel 60 F254 plate (E. Merck) developed with an acetonitrile: water (7 : 3) solvent. The purity and charge sign of 99mTc-PMIDA was determined by paper electrophoresis. Paper strips (Toyo filter paper 51A) were marked at the mid-point before soaking in buffer. The strips were blotted lightly and samples applied to the mid-point from a micropipet, then transferred to the electrophoresis tank and run at a constant voltage of 600 V for 30 min with 0.1 M tris buffer, pH 7.4. The strips were removed after running, and air-dried while mounted on the stiff card to prevent curling. 
The TLC plates and paper strips were counted on the images in a gamma camera (Ohio-Nuclear Co.) equipped with a high resolution collimator and a digital computer (VP-450). Movement of paper electrophoresis was determined relative to anionic markers 99mTc-DTPA (movement = +4.6 cm) and 99mTc pertechnetate (movement = +9.0 cm). 
The partition coefficient was measured by mixing the 99mTc-PMIDA with 1 ml each of 1-octanol and 0.1 M phosphate buffer (pH 7.0) in a glass tube. This tube was shaken for 20 min at 25deg.C and then centrifuged at 1500 rpm for 10 min. Two precisely measured samples (0.1 ml each) from the 1-octanol and buffer layers were counted in a well-type gamma counter (Packard COBRA II). The partition coefficients were determined by calculating the ratio of the radioactivity of the octanol layer to that of the buffer layer. This measurement was generally repeated three times. 
Biodistribution study in mice 
The time course of organ distribution was determined in four ICR mice. Each was injected with 0.15 ml (2.7 MBq, 0.20 mg) of the 99mTC-PMIDA through the tail vein. Excretion studies of 99mTc-DTPA (2.7 MBq, 0.35 mg) and 131I-OIH (1.85 MBq) were also performed for comparison. The mice were killed by collection of blood from the heart at 5,15,30 and 60 min after injection. The organs or tissues were removed and weighed. The 99mTc radio-activity of all organs, blood and urine was counted by images in a gamma camera (Technicare Co.) to measure the radioactivity of the excreted urine and the total radio-activity. The low 99mTc radioactivity of the blood, Iiver, spleen and stomach was also counted in a well-type gamma counter. The correction of each count in two measurements was performed by coincidence with the blood radioactivity. The 131I radioactivity was counted in a GM counter. The percentages of the injected dose per organ (ID/organ) were determined by the ratio of organ radioactivity to the total radioactivity. Statistical analysis was performed by Student's t-test for unpaired data. 
Imaging study in mice and rats 
The gamma camera was a SIGMA 420, a 37 tube array of 50.8 mm bialkali PMTs coupled to a 33.6 cm diameter by 0.63 cm thick NaI (Tl) Scintillation crystal with a patient applied for Ohio-Nuclear electronic and optical techniques for ultra high resolution. Its field of view is a hexagon that is 24.8 cm across the fiats. 
Mice or rats were injected through the tail vein with 0.15 ml (2.7 MBq, 0.0009 mmol) of 99mTC-PMIDA or 99mTc-DTPA for imaging studies. Images of four mice or rats were obtained with a gamma camera (SIGMA 420) equipped with a high resolution collimator (Model 14S17033 High Sensitivity parallel hole) in the fixed position of the mice and were collected with a digital computer (Vip-450). The software was Vip-450F V5.0 System. The total count in each scintigram frame was 5 x 104. 
Blood disappearance rates 
Three male Wistar rats weighing 350 +- 20 g were used in the experiment. Each animal was anesthetized with 25 mg/kg of sodium pentobarbital intraperitoneally. A catheter with three necks was placed in a femoral vein for injection of 99mTc complex and isotonic saline or probenecid infusion. They were placed under a gamma camera provided with digital storage (VP-450). Following infusion of isotonic saline at the rate of 20 ul/min, 90 min, digital images were acquired in the anterior projection simultaneously with a bolus injection of 99mTc-PMIDA (7.4 MBq) or 99mTc-DTPA (7.4 MBq). Rapid (10 sec) serial digital images were acquired for 24 min and were used to quantitatively estimate blood clearance by the disappearance of radioactivity in the heart. After infusion of 50 mg/kg/hr probenecid18,19 at the rate of 20 ml/min, 90 min in the same animal, 99mTC-PMIDA (7.4 MBq) or 99mTc-DTPA (7.4 MBq) was injected and measured as above. 
For blood studies of 131I-OIH, a catheter was placed in a femoral vein for tracer injection and saline or probenecid infusion, and another was placed in a femoral artery for blood sampling. Following infusion of saline at the rate 20 ul/min, 90 min, 1.85 MBq of OIH was injected, and 0.1-0.2 ml samples of blood were taken at 1,5,10,15 and 20 min after the injection. After infusion of 50 mg/kg/hr probenecid at the rate of 20 ul/min, 90 min in the same animal, 1.85 MBq of OIH was injected and the effect of the administration of probenecid on the rate of blood clearance was investigated. The blood samples were counted in a well-type gamma counter. The correction of each radioactivity between 99mTc and 131I was performed by coincidence with the radioactivity at 1 min. The rate constant of blood clearance was calculated from 99mTc radioactivity or 131I radioactivity from 5 min to 20 min. 
Glycerol-induced acute renalfailure 
Myohemoglobinuric acute renal failure in four mice20 was induced by the injection of 50% glycerol (1 ml/100 g) into the muscles of the left hind limb. One day after the glycerol injection, mice showed signs of marked oliguria and 99mTc complex solution was injected. The kidney time activity curves and cumulative percent dose in the bladder was investigated by means of a gamma camera with digital storage. 
Distribution of radioactivity in blood 
99mTc-PMIDA complex was injected into the rat through the tail vein. The blood was collected in a heparinized syringe at 1 hr after the injection. One ml of the blood was mixed slowly with 6 ml of saline solution. The mixture was layered on the top of 3 ml of Conray 400-Ficoll solution and centrifuged at 400 x g ( 1550 rpm) for 30 min. Each fraction was separated and the radioactivity measured with a well-type gamma counter. 
Measurement of in vivo plasma protein binding 
99mTc-PMIDA or 99mTc-DTPA was injected through the tail vein into each three mice. The blood was collected in a heparinized syringe at 30 min and 1 hr after the injection. Heparinized blood samples were centrifuged and the cells were removed. The method for measuring protein binding was gel filtration with a PD-10 column prepacked with Sephadex G-25.11 A 0.05 ml sample of the serum was applied to the PD- 10 column and eluted with saline. Each 0.2 ml fraction was put into separate tubes and counted in a well-type gamma counter. 
Toxicity study 
The acute toxicity of PMIDA and DTPA were deterrnined in five male ICR mice weighing 30 +- 2 g each, PMIDA and DTPA were dissolved in saline and the pH of these solutions was adjusted to 7.4 with 0.1 M NaOH. A dose of 1 g/kg 0.05-0.1 M solution of ligand was administered intravenously to each mouse. The mice were followed up for 30 days with normal animal care. 
RESULTS 
Chemical studies 
The 99mTc complex of PMIDA showed a sharp single peak, the Rf value for which fell to 0.40 with 70% acetonitrile. In this solvent pertechnetate gave an Rf value of 0.98, and reduced hydrolyzed 99mTc remained at the origin. Table 1 shows the yields of 99mTc labeling for the various concentrations of PMIDA and the various pH. As judged by the chromatography, PMIDA was complexed with 99mTc in a yield constantly greater than 99% in the pH range 3.0-8.0. PMIDA was soluble in acidic and neutral aqueous media and rarely formed precipitates or colloid with Sn in the pH range 4.0-7.5. The same chromatogram was obtained even after the product had stood for 24 hr at room temperature. On the other hand, the 99mTc labeling of N-(benzyl)iminodiacetic acid, N-(3-pyridylmethyl)-iminodiacetic acid and N-(4-pyridylmethyl)iminodiacetic acid did not give a sharp single peak. 
Electrophoresis of 99mTc-PMIDA on paper strips showed a single species which migrated about 1.2 cm toward the anode, indicating a slightly negative-charged species. In a control of hydrolyzed 99mTc there was no migration under the conditions of the experiment. Under the same conditions 99mTc-DTPA and pertechnetate migrated about 4.6 cm and 9.0 cm toward the anode, respectively. The logarithm of the octanol/water partition coefficients of the 99mTc complexes can be used to predict the relative urinary/hepatobiliary clearance of the complex.21 We selected 99mTc-E-HIDA complex as a lipophilic control complex. The log P (the radioactivity of octanol/ the radioactivity of water) of 99mTc-PMIDA, 99mTc-DTPA and 99mTc E-HIDA were 4.0,-5.0 and -1.2, respectively. 99mTc-PMIDA was a more hydrophilic complex than 99mTc-E-HIDA. 
In vivo distribution studies 
Table 2 shows the organ distribution of 99mTc-PMIDA in mice from 5 min to 1 hr. Organ distributions indicated clearance by the kidneys. The initial radioactivity of the liver and intestines is due to residual blood. The values at 30 min were 84% of the injected dose in the urine for 99mTc-PMIDA, 97% for OIH and 84% for 99mTc-DTPA. The difference between 99mTc-PMIDA and OIH was significant (p < 0.01), but the difference between 99mTc-PMIDA and 99mTc-DTPA was not. Typical scintigrams (Fig. 1 ) from the rats revealed that the 99mTc complex was rapidly excreted in urine, and provided excellent renal images with no significant extrarenal background. 
Figure 2 shows the time-activity curves for the heart in rats administered 99mTc-PMIDA, 99mTC-DTPA or 131I-OIH. The disappearance of 99mTc radioactivity from the blood (t1/2 = 11.7 +- 0.6 min) was almost same as that of 99mTc-DTPA (t1/2 = 11.8 +- 0.5 min) and lower than that of 131I-OIH (t1/2 = 9.9 +- 0.5 min). The rate of blood clearance of 99mTc-PMIDA and 99mTc-DTPA was unaffected by the administration of probenecid used as a test for tubular secretion by the weak-acid mechanism. On the other hand the rate (t1/2 = 13.6 +- 0.6 min) of blood clearance for 131I-OIH was decreased by the administration of probenecid. 
Figure 3 shows the time-activity curves for the kidneys and cumulative percent dose in the bladder in mice with myohemoglobinuric acute renal failure. The time-activity curves for the kidneys became slightly abnormal and the cumulative percent dose in the bladder was decreased. 
Radioactivity in blood 
The main radioactivity (95.27%) was found in blood plasma. The radioactivity of blood corpuscle fractions was almost completely removed in the wash. 
When the serum sample was gel-chromatographed on a PD- 10 column, plasma protein was found in fractions 17 to 23, 99mTc-PMIDA in fractions 26 to 58 and 99mTc-DTPA in fractions 25 to 55. 99mTc-DTPA had the propensity to bind to plasma protein, 3.6 +- 0.6% at 30 min and 12.8 +- 3.0% at 1 hr 99mTc-PMIDA was only bound to plasma protein, 1.4 +- 0.29% at 30 min and 3.2 +- 0.5% at 1 hr. 
Toxicity 
Toxicity has shown the minimum lethal dose of the intravenously injected PMIDA to be 60 mg/kg in mice. On the other hand, the minimum lethal dose of injected DTPA was 30 mg/kg. In the toxicity study, no animal died during the period at a dose lower than 55 mg/kg of PMIDA. No significant differences in body weight were observed between the tested animals and the controls during the 30 days after administration. 
DISCUSSION 
PMIDA, a tetradentate chelating agent, formed a stable chelate with reduced 99mTc. PMIDA was soluble in aqueous media and was labeled with 99mTc in high yield by a simple procedure. A single uniform complex was obtained over a wide pH range of the solution. These properties are favorable to kit preparation. 
The lipophilicity of the complex must also be kept within certain limits to minimize the degree of hepatobiliary excretion of the complex 99mTc-PMIDA is more hydrophilic than 99mTc-E-HIDA. This characteristic is essential in an ideal agent for measuring GFR. 
The 99mTc labeling of N-(benzyl)iminodiacetic acid, N-(3-pyridylmethyl)iminodiacetic acid and N-(4-pyridylmethyl)iminodiacetic acid did not give a sharp single peak. On the other hand, 99mTc labeling of PMIDA gave a sharp single peak. In an attempt to explain the relationship between the ligand structures, these results show that the PMIDA: Tc ratio in the complex is 1 : 1 and not 2 : 1. The position of the pyridine nitrogen atom and the iminodiacetate group on the PMIDA ligand may play an important role in this complex. It seems that the coordination sphere includes two nitrogens and two oxygens of these carboxylates.
Agents excreted by glomerular filtration must be freely filterable by the glomerular ultrafilter. The ideal agent for measuring GFR must not be bound to plasma proteins or to other blood components either reversibly or irreversibly.22 Plasma analysis by gel filtration showed that 99mTc-PMIDA was only bound to plasma proteins 1.4% at 30 min and 3.2% at 1 hr. Though 99mTc-PMIDA seen to be superior to 99mTc-DTPA in the results of plasma analysis, this difference was within the limits of error in results for distribution. As the chelating ability of PMIDA was lower than that of DTPA, we expected PMIDA to impair renal function much less. 
99mTc-PMIDA has excellent renal excretion characteristics, as confirmed by the organ distribution studies in animals. Scintigraphic study of 99mTc-PMIDA suggests its potential utility for assessment of the renal system. The rate of blood clearance was unaffected by the administration of probenecid used as a test for tubular secretion by the weak-acid mechanism. This result shows that 99mTc-PMIDA is not actively or passively reabsorbed or secreted by the renal tubular epithelium. 
The results for animals with acute myohemoglobinuric renal failure suggest that 99mTc-PMIDA may be a useful renal function radiopharmaceutical. These results are quite satisfactory for a renal functional agent. 
ACKNOWLEDGMENT 
This work was supported in part by funds from the Central Research Institute of Fukuoka University. 
REFERENCES 
1. Richards P, Atkins HL. Technetium-99m-labeled compounds. J Nucl Med 7: 165-170, 1967. 
2. Hosain F, Reba RC, Wagner Jr HN. Measurement of glomerular filtration rate using chelated ytterbium- 169. Int J Appl Radiat lsot 20: 517-521 , 1969. 
3. Bianchi C. Measurement of the glomerular filtration rate. Progr Nucl Med 2: 21-53, 1972. 
4. Skov PE. Glomerular filtration rate in patients with severe and very severe renal insufficiency. Determined by simultaneous inulin, creatinine and I-125-iothalamate clearance. Acto Med Scand 187: 419-428, 1970. 
5. Russell CD, Speiser AG. Iminodiacetate complexes of technetium: An electrochemical study. Int J Appl Radiat Isot 33: 903-906, 1982. 
6. Agha N, Persson RBR. Comparative labelling and biokinetic studies of 99mTC-EDTA (Sn) and 99mTc-DTPA (Sn). J Nucl Med 16: 30-35, 1977. 
7. Dewanjee MK, Brueggeman PM. Dissociation constants of Tc-99m chelates with serum protein. J Nucl Med 18: 625, 1977. 
8. Levin VI, Gracheva MA, Ilyushchenko ON. The stability constant of 99mTc-DTPA complex. Int J Appl Radiat Isot 31 : 382-385, 1980. 
9. Russell CD, Crittenden RC, Cash AG. Determination of net ionic charge on Tc-99m-DTPA and Tc-99rn-EDTA by a column ion-exchange method. J Nucl Med 21 : 354-360, 1980. 
10. Hauser W, Atkins HL, Nelson KG, Richard P. Technetium-99m-DTPA: A new radiopharmaceutical for brain and kidney scanning. Radiology 94: 679-684, 1970. 
11. Russell CD, Bischoff PG, Rowell KL. Kontzen F, Lloyd LK. Tauxe WN, et al. Quality control of Tc-99m DTPA for measurement of glomerular filtration. J Nucl Med 24: 722-727, 1983. 
12. Carlsen JE, Mu'ller ML, Lund JO, Trap-Jensen J. Comparison of four commercial Tc-99m (Sn)-DTPA preparations used for the measurement of glomerular filtration rate. J Nucl Med 21 : 126-129, 1980. 
13. Noll B, Seifert S, Munze R. Preparation and characterization of technetium (IV) complexes with diethylenetriaminepentaacetic acid and ethylenediaminetetraacetic acid as ligands. Int J Appl Radiat lsot 34: 581-584, 1983. 
14. Trump BF, Berezesky IK, Smith MW, Phelps PC, Elliget KA. The relationship between cellular ion deregulation and acute and chronic toxicity. Toxicol Appl Pharmacol 97: 6-22, 1989. 
15. Davison A, Sohn M, Orvig C. Jones AG, LaTegola MR. A tetradentate ligand designed specifically to coordinate technetiumn. J Nucl Med 20: 641, 1979. 
16. Davison A, Jones AG, Orvig C. Sohn M. A new class of oxotechnetium (5+) chelate complexes containing a TcON2S2 core. Inorg Chem 20: 1629-1632, 1980. 
17. Irving H, DaSilva JJRF. Metal complexes of N-(2-pyridylmethyl)iminodiacetic acid. J Chem Soc 1963: 945-952, 1963. 
18. Fritzberg AR. Kashima S. Eshima D, Johnson DL. Synthesis and biological evaluation of technetium-99m MAG3 as a hippuran replacement. J Nucl Med 27: 111-116, 1986. 
19. Klopper JF. Hauser W, Atkins HL. Eckelman WC. Richards P. Evaluation of 99mTc-DTPA for the measurement of glomerular filtration rate. J Nucl Med 13: 107-110, 1972. 
20. Ayer G, Grandchamp A, Wyler T, Truniger B. Intrarenal hemodynamics in glycerol-induced myohemoglobinuric acute renal failure in the rat. Circulation Research 29: 128-135, 1971. 
21 . Burns HD, Worley P, Wagner Jr HN, Marzilli L, Risch V. Design of technetium radiopharmaceuticals. In The Chemistry of Radiopharmaceuticals. Heindel ND. Burns HD, Honda T, Brady LW (eds.), New York. Masson Publishing USA. Inc., pp. 269-289, 1978. 
22. Russell CD. Radiopharmaceuticals used to assess kidney function and structure. In Nuclear Medicine in Clinical Urology and Nephrology. Tauxe WN, Dubovsky EV (eds.), Appleton-Century-Crofts, Norwalk, Connecticut, pp.7-29, 1985.