ORIGINAL ARTICLE Annals of Nuclear Medicine Vol.10, No.4, 375-381, l996 Comparison of cationic myocardial perfusion agents: Characteristics of accumulation in cultured smooth muscle cells Kayoko NAKAMURA, Toshikazu SAMMIYA, Jun HASHIMOTO, Ryochi ISHIBASHI, Kazuhiro MATSUMOTO and Atsushi KUBO Department of Radiology, Keio University School of Medicine The uptake and washout kinetics of two cationic lipophilic 99mTc-labeled myocardial perfusion agents, 99mTc-methoxyisobutylisonitrile (99mTc-MIBI) and 99mTc-1,2-bis[bis-(2-ethoxyethyl)-phosphino]ethane (99mTc-Tetrofosmin), were studied in cultured smooth muscle cells and compared to the conventional myocardial perfusion agent, 201Tl. Both 99mTc-MIBI and 99mTc-Tetrofosmin had a 4-fold greater uptake than 201Tl, and they were washed out of cells through similar kinetics which had slower rates than 201Tl. Incubation with metabolism inhibitors had a modest influence on the uptake of these two 99mTc-labeled agents, although their extent and inhibited sites were slightly different. Ion transport inhibitors did not affect the uptake of 99mTc-MIBI, although the 99mTc-Tetrofosmin uptake was slightly inhibited when the Ca2+ channel was blocked. Our studies indicate that 99mTc-MIBI and 99mTc-Tetrofosmin were taken up by smooth muscle cells in similar pharmacokinetic patterns, but their accumulation reflected a different meaning for cell viability. Key words: 99mTc-MIBI; 99mTc-tetrofosmin; 201Tl, smooth muscle cells INTRODUCTION NEW 99mTC-LABELED myocardial perfusion agents, which may replace 201Tl, have been introduced during the last few years. Among them, 99mTc-methoxyisobutylisonitrile (99mTc-MIBI) and 99mTc-1,2-bis[bis(2-ethoxyethyl)-phosphino]ethane (99mTc-Tetrofosmin), are both potential for routine clinical studies of myocardial perfusion. 99mTc-MIBI is one of the cationic isonitrile 99mTc-complexes; that is, it has a net charge of +1 and a coordination number of six with six isonitrile ligand groups. On the other hand, 99mTc-Tetrofosmin is a lipophilic complex with two diphosphone ligands surrounding a technetium dioxo core; it too has a + 1 charge. Several laboratories, including ours, evaluated the biodistribution of both 99mTc-agents in normal volunteers.1,2 It is interesting that both of them showed similar heart uptake and retention and blood clearance kinetics, in spite of their different chemical structures. Extensive studies have been carried out to evaluate the detection of myocardial disease by either of these two 99mTc-labeled agents. There have been no significant differences observed between the two agents. On the other hand, the uptake mechanism for either of the two 99mTc-labeled compounds remains unclear. By using erythrocytes,3 myocyte cells,3-9 and tumor cells,10-16 the accumulation mechanism of 99mTc-MIBI was exclusively studied. It was also clinically reported that 99mTc-MIBI localizes in several tumors in patients.17 On the other hand, there have been few reports estimating the accumulation mechanism of 99mTc-Tetrofosmin in cells,18 while the comparison of 99mTc-MIBI with other 99mTc-labeled compounds has been studied in some tumor cells.11,12 It is important to clarify the mechanism of uptake of myocardial perfusion agents to interpret the biochemical and physiological characteristics of tumors as well as heart disease and to compare 99mTc-labeled compounds with regard to these points. The uptake of 99mTc-labeled agents in the heart involves many factors such as blood flow, vascularity, plasma proteins, and so on. To compare the two 99mTc-labeled agents equivalently, we need a very simple in vitro system such as a cell culture model. Myocytes are generally used in most experiments, but they have to be prepared for each@experiment, and their biochemical and physiological characteristics are not always uniform. Normalization of the cell characteristics is necessary to compare the two labeled compounds. The cultured cells have homogeneous characteristics, but the establishment of a normal cell line is not easy. Recently smooth muscle cells isolated from Caucasian blood vessels have been able to be cultured in a monolayer for a few generations under definitive conditions and they have been commercially available. The cultured normal cell is an adequate and simple model to use for comparing the two 99mTc-labeled agents and 201Tl, although the results are not directly comparable with those obtained in myocytes. This paper deals with comparative studies of the three myocardial perfusion agents, 201Tl, 99mTc-MIBI, and 99mTc-Tetrofosmin, with regard to their uptake and washout pharmacokinetics. We also investigated the effect of some metabolism and ion transport inhibitors on the uptake of 201Tl and 99mTc-labeled compounds. The purpose of this study was to differentiate between the two 99mTc-labeled agents in an in vitro system, which differmence has not been observed clinically. MATERIALS AND METHODS 99mTc-Tetrofosmin (99mTc-l,2-bis[bis(2-ethoxyethyl)-phosphino]ethane) This ligand was formulated into a freeze-dried kit. The agent was prepared from a freeze-dried kit (Myoview, Amersham International plc) by reconstitution with ap-proximately 3 ml of a sterile sodium pertechnetate solution containing 190-370 MBq of 99mTc. The vial was shaken gently to ensure complete dissolution of the lyophilized powder, and the solution was allowed to stand at room temperature for 15 minutes. 99mTc-MIBI (99mTc-methoxyisobutylisonitrile) This agent was prepared from a freeze-dried kit (Cardiolite, Daiichi Radioisotopes Inc.) by reconstruction with approximately 3 ml of a sterile sodium pertechnetate solution containing 190-370 MBq of 99mTc. The vial was heated at 100degC for 10 min and allowed to cool to room temperature. All preparations were tested by thin-layer chromatography for quality control according to the manufacturer's recommendations and were used within 2 hours of reconstitution. The radiochemical purities of 99mTc-Tetrofosmin and 99mTc-MIBI ranged from 95 to 99%. 201Tl was obtained as a sterile, pyrogen free, clinically injectable radiopharmaceutical from Amersham International plc. Smooth cell-AO; Aortic Smooth Muscle Cells (AOSMC 751 3deg) These cells were purchased from Clonetics, Inc. through Kurabo, Inc. Primary cells were isolated from a male Caucasian on 03/27/92. The cells are continually observed and evaluated for characteristic smooth muscle cell morphology. The cells were maintained in a stationary phase in a Normal Human Smooth Muscle Cells Growth Medium (S-BM) medium supplemented with 5% fetal calf serum, human endothelial growth factor (EGF), recombinant fibroblast growth factor (r-FGF-B) and antibiotics. Cellular uptake and retention of myocardial perfusion agents Cells harvested from the culture were suspended in S-BM medium containing 5% of fetal calf serum (incubation buffer) with a concentration of approximately 10E6 cells/ml. Cell suspensions (500 micro l) were incubated with 50 micro l of 201Tl 99mTc-Tetrofosmm or 99mTc-MIBI (c.a. 3.7 KBq/ ml) in the 1.5-ml tubes for various times at 37degC. Measured samples of the incubation buffer were withdrawn to standardize the cellular data to the concentration of extra-cellular activity. The incubation buffer was completely removed from the tube by centrifugation followed by rinsing the cells twice with cold fresh incubation buffer to clear the extracellular spaces. The radioactivities of the aliquots of cell suspension in the tubes were then counted. The nonspecific binding of radioactivity to the blank tube, which was pretreated with 1% BSA (bovine serum albumin)/PBS (phosphate bufferized saline), was 10-15% and 2% of the added activity for 99mTc-labeled agents and for 201Tl, respectively. The results are expressed as the radioactivities of 201Tl and 99mTc per cell and are normalized to a constant incubating solution concentration. This quantification allowed a direct comparison of data for the various experiments and cultures. Cellular washout Cellular washout experiments were cohducted by first incubating the cells for one hour at 37degC in incubation buffer containing 201Tl, 99mTc-Tetrofosmin or 99mTc-MIBI. For stepwise washout experiments, the cells were rinsed with 100 micro l of incubation buffer and the radioactivities of the cell pellets were determined after spinning the cell suspensions. This procedure was repeated three times. For the washout-kinetic studies, the cells were twice rinsed consecutively in 100 mmicro l of ice-cold incubation buffer and incubated in fresh incubation buffer at 37degC for various lengths of time. The cells were processed as above to quantify the retained activity. The data values represent the mean for four or more individual observations. The error bars on the graphs were not shown when the SEM did not exceed 15% of the mean value. Effects of inhibitors on the uptake of myocardial perfusion agents Tests for the effects of metabolism inhibitors were performed by preincubating cells at 37degC with the drug for 30 min followed by a 30 min 201Tl, 99mTc-Tetrofosmin, or 99mTc-MIBI uptake. The following inhibitors were tested: rotenone ( 10 micro M; inhibition of electron transfer); dinitrophenol (DNP, 10 micro M; uncoupiing of phosphorylation from electron transfer); and iodoacetate (IAA, 1 mM; inhibition of glycolysis). The following cationic membrane transport inhibitors were also evaluated for a possible effect on the uptake of the perfusion agents in this model: ouabain (lO micro M; inhibition of Na+ /K+ ATPase activity); amiloride (100 micro M; blocking a Na+/H+ exchange); and verapamil ( 1 micro M; blocking a Ca2+ channel). The effects on the uptake were monitored by exposing the cells to the inhibitors for 30 min followed by a 30-min isotope incubation in the presence of the drug. The cellular viability was visually examined by the trypan blue dye exclusion technique. The results, in bar graph form, are expressed as the pooled mean for four individual tubes. RESULTS Cellular uptake and retention of myocardial perfusion agents Incubation of cultured smooth muscle cells with one of the three agents tested at 37degC resulted in an exponential accumulation of activity up to an apparent plateau or equilibrium condition. After normalizing the results to a uniform extracellular loading-solution concentration, a comparison of time-dependent uptake could be made for the different agents. 201Tl and 99mTc-labeled agents were accumulated in smooth muscle cells at a different rate and to a distinctly different plateau value (Fig. 1 ). The data gave a good fit to the biexponential curve. Note that extraction of 201Tl within the first few minutes is almost equal for the 99mTc-labeled agents, but at later times 99mTc-labeled agents were accumulated to a greater extent. The maximum uptake of 99mTc-Tetrofosmin was almost 4-fold greater than that of 201Tl and higher ratios persisted for accumulation times greater than 3 hours. Kinetic comparisons among the three agents are summarized in Table l. Cellular washout After incubation to equilibrium, the cells were incubated in isotope-free incubation buffer. Figure 2 compares the three agents with respect to the stepwise washouts with incubation buffer. The first washout was due, at least in part, to the loose extracellular matrix. 99mTc-Tetrofosmin was washed out by the first rinsing with 65% of the initial apparent uptake value, in spite of 201Tl only being washed out at 35%. Only a small amount of 99mTc-Tetrofosmin was washed out by the next, second and third rinsing steps, while 99mTc-MIBI and 201Tl were released from the cells step by step. The cells were rinsed with cold incubation bu er twice, and then they were incubated with fresh incubation buffer at various times at 37degC (Fig. 3). Analysis of the washout curve suggested that the process may be biexponential with two compartments with a rapid initial phase, and a slower secondary component that was released continuously to 120 min of incubation. It was interesting that 17% of the 99mTc-Tetrofosmin and 15% of the 99mTc-MIBI remained in the cells at 120 min, while 96% of the 201Tl was washed out of the cells. Compansons of the washout kinetics of these three agents are summarized in Table 2. The release of the 99mTc-labeled agents was slower than that of 201Tl. Effects of inhibitors on the uptake of myocardial perfusion agents To determine if the cellular accumulation of these myocardial perfusion agents occurred by a similar mechanism, their uptake was monitored after chemically inhibiting selective metabolic pathways and ion transport. Conditions were chosen that would produce the least effect on the smooth muscle cells. Estimates of cellular viability by the trypan blue exclusion method showed a small effect after 90 min exposure to the inhibitor. From the cell uptake results shown in Fig. 1 , the 30 min-incubation with 201Tl or 99mTc-agents followed by the preincubation with inhibitors for 30 min may be enough to reach the plateau level. Following 30 min of preincubation in various metabolism inhibitors, 201Tl did not demonstrate any effects. This behavior is in striking contrast to that of 99mTc-labeled agents. Figure 4 shows that DNP decreased the 99mTc-Tetrofosmin net uptake, while IAA decreased the 99mTc-MIBI net uptake (p < 0.01). To test for linkage to a specific transport system, uptake experiments on the three myocardial perfusion agents were performed in the presence of three different ion transport inhibitors (Fig. 4). A significant decrease (p < 0.001) occurred in the 201Tl uptake with ouabain. The uptake of 99mTc-MIBI was not decreased by any ion transport inhibitors we investigated. On the other hand, verapamil inhibited the accumulation of 99mTc-Tetrofosmin slightly (p < 0.05). These effects were observed in a similar manner when the incubation medium did not contain growth factors or sodium bicarbonate. DISCUSSION Both 99mTc-Tetrofosmin and 99mTc-MIBI hold promise as potential myocardial perfusion 99mTc-labeled agents. Clinically they have behaved in a very similar manner although their accumulation and retention mechanism have remained obscure. We compared the characteristics of two 99mTc-labeled agents by using cultured smooth muscle cells. 201Tl, a conventional myocardial perfusion agent, was also studied as a reference to compare with the two 99*Tc-labeled agents. The smooth muscle cells which we used were isolated from human aortic smooth muscle and could be maintained in a stationary phase for several generations. Myocytes were generally isolated from some experimental animals for every experiment. Cultured smooth muscle cells can be expected to have the same biochemical and physiological characteristics in any experiment. This led us to use the cultured smooth muscle cells as biological material to compare these three myocardial perfusion agents although smooth muscle cells are quite different from myocardial cells with regard to biochemical properties. Both 99mTc-Tetrofosmin and 99mTc-MIBI are lipophilic and have a positive charge, which causes them to stick to the surface of plastic tubes. Referring to the report from Amersham plc (personal communication), we pretreated the tubes with 1% BSA/PBS. We noticed that l0-15% of the radioactivity was still stuck to the surface of the pretreated tubes. Because the non-specific adsorptions were much dependent on the incubation medium including inhibitors, we did not evaluate any differences with less than 15% deviation. Both 99mTc-Tetrofosmin and 99mTc-MIBI exhibited very similar uptake kinetics, which were significantly different from 201Tl. Both of the 99mTc-labeled agents were accumulated in the cell at a rate slower than 201Tl in the early phase, but they took a little longer to reach the plateau than 201Tl did. Consequently, the plateau levels of the 99mTc-labeled agents were four times that of 201Tl. The washout kinetics of 99mTc-labeled agents were also found to have similar patterns. Their washout rates were generally lower than those of 201Tl. It is interesting that 99mTc-labeled agents still remained in the cell even after a l20 min-incubation period with fresh incubation buffer. A still more interesting observation was that 99mTc-Tetrofosmin was released from cells once, while 99mTc-MIBI and 201TI were washed out step-by-step by the fresh isotope-free buffer. It is well known that 201Tl is washed out of the heart and redistributes again. Clinically it was also observed that very small amounts of 99mTc-labeled agents are washed out of the heart.2 Our previous clinical comparison of the two 99mTc-labeled agents showed that 99mTc-MIBI appeared to be washed out of the heart more rapidly than 99mTc-Tetrofosmin,1,19 but their precise pharmacokinetic values remain unclear. These results suggest that both of the 99mTc-labeled agents are retained in the cell in a different manner, while 201Tl exists loosely in the cell similar to K+. How 99mTc-labeled agents are accumulated in the cell was also evaluated on the basis of the effect of chemical inhibitors on net uptakes. The effect of inhibitors on the 99mTc-MIBI uptake in ventricular chick myocytes was reported by Kronauge JF et al.9 They showed that IAA and CCCP inhibited the uptake, but no effect was observed in the presence of DNP, which is an uncoupler of phosphorylation like CCCP, in our experiments. Platts EA et al. also reported the effect of cation channel and metabolism inhibitors on the uptake of 99mTc-Tetrofosmin in rat myocytes.18 They showed that cation channel inhibitors, such as 100 micro M of ouabain, 10 micro M of bumetanide and l,000 micro M of amiloride, did not have any significant effect. Their report on the metabolic inhibition was partially inconsistent with our experimental results. They showed that CCCP inhibited cell uptake, indicating that 99mTc-Tetrofosmin was retained in the cell principally through the mitochondrial membrane potential. Our experimental results showed that mitochondrial electron transport inhibitors, such as rotenone, as well as DNP, uncoupler of phosphorylation, affected the cell uptake, suggesting that 99mTc-Tetrofosmin is taken up in smooth muscle cells partially by the mitochondrial electron trans-port energy and partially by its membrane potential. The effect of IAA on the 99mTc-Tetrofosmin net uptake was more significant in their myocytes than in our smooth muscle cells. This discrepancy may be due to the different properties of the cells that were used in the two studies: myocardial cells contain a greater density of mitochondria than smooth muscle cells. The net uptake of 99mTc-Tetrofosmin in smooth muscle cells in our experiments was approximately l/lOO of that in myocytes in Platts' report.18 Involvement of mitochondrial energy in the transport system would also be different from cell type to cell type, but the precise reason is unclear. As far as the smooth muscle cells we used are concerned, 99mTc-Tetrofosmin is taken up partially through the mitochondria by way of mitochondrial site electron transport and its membrane potential. On the other hand, the uptake of 99mTc-MIBI in the cells involves mitochondrial electron transport potentials rather than its membrane ones. The mitochondrial function plays an important role in the uptake and retention of both 99mTc-labeled agents as other reports have described, and the extent of its role was greater with 99mTc-Tetrofosmin than with 99mTc-MIBI. Furthermore, 99mTc-MIBI uptake would be more sensitive to cell glycolysis than that of 99mTc-Tetrofosmin. Considering our experimental results together with other reports9,18 we assume the uptake and retention mechanism of the 99mTc-labeled agents in smooth muscle cells to be as follows: (1) 99mTc-Tetrofosmin is taken up by both mitochondrial electron transport systems and membrane potential. It is also affected by the calcium ion channel. It is partially held in the mitochondrial fraction with relatively strong affinity. (2) 99mTc-MIBI is taken up mainly by the mitochondrial electron transport potential to a lesser extent than 99mTc-Tetrofosmin. Its retention in the cell is relatively loose. These results suggest that 99mTc-Tetrofosmin can predict a mitochondrial viability unlike that with 201Tl. On the other hand, 99mTc-MIBI uptake could be used to predict the density of mitochondria in cells rather than the mitochondrial and cell viabilities. 99mTc-Tetrofosmin is retained in the cell with a stronger affinity than 99mTc-MIBI and 201Tl. Consequently, 99mTc-MIBI and 99mTc-Tetrofosmin are taken up by the cell in a similar pharmacokinetic pattern, but their accumulation reflects a different form of cell viability. THese assumptions are based on our experimental model using an established smooth cell line with homogeneous characteristics. They would not work for heart tissue which contains a higher density of mitochondria, or for a tumor of which the characteristics are far different from those of the normal cell. Furthermore, generally speaking, it is very difficult to correlate the experimental cell results in vitro with the clinical behavior of myocardial perfusion agents. 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