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(Circulation. 1997;96:2325-2331.)
© 1997 American Heart Association, Inc.

Articles

Myocardial Uptake and Redistribution of ^99mTc-N-NOET in Dogs With Either Sustained Coronary Low Flow or Transient Coronary Occlusion

Comparison With ²⁰¹Tl and Myocardial Blood Flow

Gérald Vanzetto, MD; Dennis A. Calnon, MD; Mirta Ruiz, MD; Denny D. Watson, PhD; Roberto Pasqualini, PhD; George A. Beller, MD; ; David K. Glover, ME

From the Experimental Cardiology Laboratory, University of Virginia, Charlottesville, and CIS-Bio International, Gif sur Yvette, France (R.P.).

	Abstract

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Background ^99mTc-N-NOET (NOET) is a new myocardial perfusion imaging agent that redistributes over time. We sought to better define the redistribution kinetics of NOET using open-chest canine models of sustained low coronary flow (protocol 1) and transient coronary occlusion followed by reflow (protocol 2).

Methods and Results In protocol 1 (n=10), NOET and ²⁰¹Tl were injected during low flow in the left anterior descending coronary artery (LAD) that was sustained for 2 hours. Protocol 2 dogs (n=6) were injected with NOET during 20 minutes of LAD occlusion followed by 2 hours of reflow. In both protocols, serial NOET planar images were acquired, and myocardial flow and 2-hour tracer activities were determined by gamma-well counting. Defect resolution was observed on images in both protocols. Initial defect count ratios, reflecting flow disparity at injection (0.66±0.03 and 0.57±0.04, respectively), increased over 2 hours (0.73±0.02 and 0.75±0.04, respectively; P<.001 versus initial). Quantitative imaging showed that NOET redistribution resulted from greater clearance from normal areas versus low-flow or transiently occluded areas. In protocol 1, 2-hour NOET and ²⁰¹Tl stenotic-to-normal tissue activity ratios were similar (0.76±0.06 versus 0.73±0.04, P=NS) and higher than injection flow ratios (0.52±0.06 and 0.56±0.07, respectively, P<.001), consistent with tracer redistribution. In protocol 2, NOET redistributed to an even greater extent (injection flow ratio, 0.27±0.04; 2-hour tissue activity ratio, 0.84±0.03, P<.001).

Conclusions NOET is the first ^99mTc-labeled myocardial imaging agent with kinetics similar to ²⁰¹Tl in experimental models, permitting redistribution imaging. NOET appears to be a promising agent for assessing patients with coronary artery disease.

Key Words: radioisotopes • ischemia • thallium • technetium

	Introduction

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To date, the quest for an optimal myocardial perfusion imaging agent that combines the radiophysical properties of ^99mTc with the redistribution kinetics of ²⁰¹Tl has largely been unsuccessful. ^99mTc-sestamibi, the most widely used ^99mTc-labeled imaging agent, permits high-quality imaging, but because it exhibits minimal redistribution over time, assessment of coronary artery disease requires two separate injections.^{1 2 3 4 5 6 7 8 9}

^99mTc-N-NOET is a new neutral, lipophilic ^99mTc-labeled myocardial perfusion imaging agent.^{10 11 99m}Tc-N-NOET has a high first-pass extraction fraction, its myocardial uptake correlates with coronary blood flow, and it exhibits significant redistribution when injected during dipyridamole stress in canine models of coronary stenosis.¹² Preliminary data suggest that ^99mTc-N-NOET myocardial perfusion scans provide diagnostic information comparable to ²⁰¹Tl in patients undergoing exercise testing for assessment of coronary artery disease.¹³ Furthermore, delayed normalization of initial ^99mTc-N-NOET defects has been shown to be correlated with ²⁰¹Tl redistribution imaging and was interpreted as ^99mTc-N-NOET redistribution.¹³

Although these earlier animal and clinical studies strongly suggest that ^99mTc-N-NOET redistribution does indeed occur under some conditions, to date there has been no carefully controlled experimental study that has examined ^99mTc-N-NOET redistribution kinetics. Accordingly, the aims of this study were (1) to better define ^99mTc-N-NOET redistribution kinetics by simultaneously comparing the myocardial uptake and redistribution of ^99mTc-N-NOET with those of ²⁰¹Tl in a canine model of sustained coronary artery low flow and (2) to assess the redistribution kinetics of ⁹⁹Tc-N-NOET in a canine model of transient coronary artery occlusion followed by early reflow. The sustained-low-flow model was selected because it is the model most frequently used to assess the ability of a tracer to redistribute (eg, ²⁰¹Tl) during prolonged hypoperfusion and consequent asynergy when flow is unchanged from the time of tracer administration to the end of the "redistribution" period. Although the second model, in which ^99mTc-N-NOET was injected during transient coronary occlusion followed by early reflow, does not have a direct clinical correlate, it is relevant because it optimizes the potential for redistribution of a tracer to occur and thus can give insights into mechanisms. This was the model used 20 years ago to define ²⁰¹Tl redistribution kinetics.¹⁴

	Methods

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Surgical Preparation
Sixteen fasted male adult mongrel dogs were anesthetized with sodium pentobarbital (30 mg/kg) and prepared as previously described.⁷ A left thoracotomy was performed at the level of the fifth intercostal space, and the heart was suspended in a pericardial cradle. A flare-type catheter was introduced into the left atrial appendage for continuous left atrial pressure monitoring and for injection of radiolabeled microspheres. An 1.5-cm section of the LAD was dissected and loosely encircled with a snare occluder that was used to maintain the LAD stenosis (protocol 1) or to set the transient LAD occlusion (protocol 2). An ultrasonic flow probe (T206, Transonic Systems) was placed around the LAD distal to the occluder. In protocol 1, a similar ultrasonic flow probe was placed around the LCx. To measure regional left ventricular systolic wall thickening, a sonomicrometer crystal (Crystal Biotech) was sutured to the epicardial surface of the heart in the central region supplied by the LAD. In protocol 1, a crystal was also placed in the LCx region. Throughout each experiment, heart rate, systemic arterial pressure, left atrial pressure, left ventricular pressure, dP/dt, LAD flow, and LAD regional systolic wall thickening were continuously recorded on a 12-channel thermal array strip-chart recorder (K2G, Astromed Inc) and simultaneously digitized and stored in real time onto a 133-MHz Pentium IBM-compatible personal computer equipped with an optical drive.

All experiments were performed with the approval of the University of Virginia Animal Research Committee and were in compliance with the position of the American Heart Association on the use of research animals.

Experimental Protocols
Protocol 1: Comparison of ²⁰¹Tl and ^99mTc-N-NOET During 2 Hours of Sustained Coronary Artery Low Flow
Experiments were performed in a canine model (n=10) of sustained low flow, and the protocol is summarized in the top panel of Fig 1. Radiolabeled microspheres were injected during baseline for the determination of myocardial blood flow. The LAD was then partially occluded to decrease ultrasonic flow by 50%. Fifteen minutes later, 18.5 MBq (0.5 mCi) of ²⁰¹Tl and a set of microspheres were injected simultaneously. A ²⁰¹Tl image was acquired 5 minutes later over a period of 4 minutes. Immediately after the end of the acquisition, 296 MBq (8 mCi) of ^99mTc-N-NOET was injected simultaneously with microspheres. Serial planar ^99mTc-N-NOET images were acquired 15, 30, 60, 90, and 120 minutes after injection. Before the dogs were killed with an overdose of potassium chloride and sodium pentobarbital, the LAD was totally occluded and 20 mL of monastral blue dye was rapidly injected into the left atrial catheter to delineate the anatomic risk area.

View larger version (17K): [in this window] [in a new window]   Figure 1. Experimental design for protocols 1 and 2. Serial in vivo 99mTc-N-NOET images were acquired 15, 30, 60, 90, and 120 minutes after 99mTc-N-NOET injection during sustained low flow (protocol 1) or after reperfusion of a transient occlusion (protocol 2). Spheres indicates radiolabeled microspheres.

Protocol 2: Myocardial Kinetics of ^99mTc-N-NOET Injected During 15 Minutes of Total LAD Occlusion Followed by 120 Minutes of Reflow
Six dogs were prepared as in protocol 1 (bottom panel, Fig 1). After the injection of baseline microspheres, the LAD was completely occluded. Two minutes later, 296 MBq (8 mCi) of ^99mTc-N-NOET and a second set of microspheres were simultaneously injected. An initial ^99mTc-N-NOET image was acquired 15 minutes later, and the LAD was then slowly reperfused over 10 minutes to restore baseline blood flow with limited hyperemia. After completion of reflow, a third set of microspheres was injected, followed by serial ^99mTc-N-NOET imaging at 15, 30, 60, 90, and 120 minutes after reflow. Serial 1-mL arterial blood samples were also collected for measurement of ^99mTc-N-NOET blood activity.

Preparation and Quality Control of ^99mTc-N-NOET
^99mTc-N-NOET kits were obtained from CIS-Bio International (Gif sur Yvette, France). Sodium ^99mTc-N-pertechnetate (740 MBq [20 mCi]) was introduced into a vial containing tin chloride dihydrate (0.02 mg), 1,2-diaminopropane-N,N,N',N'-tetraacetic acid (5.00 mg), succinyl dihydrazine (10.00 mg), and sodium phosphate buffer (pH 7.8). After 15 minutes at room temperature, 1 mL of a solution containing N-ethoxy-N-ethyl dithiocarbamate of sodium monohydrate (10.00 mg/mL) and 1 mL of a solution of dimethyl-ß-cyclodextrine (10.00 mg/mL) were added to the first vial. Ten minutes later, ^99mTc-N-NOET was ready to be injected. Quality control was performed with thin-layer chromatography with Silicagel plates (Gelman Sciences) and dichloromethane. Radiochemical purity was >90% in each experiment.

Microsphere-Derived Regional Myocardial Blood Flow, Quantification of Tracer Uptake, and ^99mTc-N-NOET Blood Kinetics
The technique used in our laboratory to quantify myocardial blood flow by radiolabeled microspheres in 72 myocardial segments from normal and stenotic regions has been described previously.^{15 16} The myocardial segments were counted for ^99mTc activities in a gamma-well scintillation counter (MINAXI 5550, Packard Instruments) within 24 hours. In protocol 1, the myocardial segments were counted for ²⁰¹Tl activity after 48 hours, when the ^99mTc had decayed. Finally, a third count was performed for myocardial flow determination 3 weeks later, when ²⁰¹Tl activity was negligible. The gamma counter window settings were ¹¹³Sn, 340 to 440 keV; ¹⁰³Ru, 450 to 550 keV; ⁹⁵Nb, 640 to 840 keV; and ⁴⁶Sc, 842 to 1300 keV. To compare regional ²⁰¹Tl and ^99mTc-N-NOET tissue activities with microsphere flow, the data from the 72 myocardial segments were expressed as stenotic-to-normal activity and flow ratios. Hypoperfused segments were defined as those in which flow at the time of tracer injection was <75% of baseline, whereas normal segments had flow >75% baseline.

In Vivo Image Acquisition and Defect Magnitude Quantification
Planar ²⁰¹Tl and ^99mTc-N-NOET images were acquired in the left lateral projection with a standard gamma camera (Technicare 420, Ohio Nuclear) equipped with an all-purpose, low-to-medium-energy collimator as previously described.¹⁶ Background was subtracted from all images by use of a previously validated algorithm^{17 18} without thresholding or filtering. To quantify the ²⁰¹Tl and ^99mTc-N-NOET images, ROIs were drawn on the defect area of the anteroseptal wall supplied by the LAD and on the normally perfused posterior wall supplied by the LCx. The LAD-to-LCx imaging defect count ratio was computed by dividing the average counts in the ischemic ROI by the average counts in the nonischemic ROI. Time-activity curves were plotted by use of the activities obtained from the ROIs on serial images, and myocardial fractional clearance of ^99mTc-N-NOET in the LAD and LCx ROIs was calculated as [(initial mean activity per pixel-final mean activity per pixel)/initial mean activity per pixel]x100.

Postmortem Determination of Risk Area and Infarct Size
The endocardial and epicardial surfaces of each heart slice and the borders of the monastral blue dye–determined risk area were traced on acetate sheets. The heart slices were then incubated for 10 minutes at 37°C in a 2% solution of TTC to delineate infarct area, and the infarct area was traced onto the previous acetate sheets. Risk and infarct areas were determined with a digital planimeter program as previously described.¹⁹

Statistical Analysis
Mean and SEM computations were performed with SYSTAT software (SPSS, Inc). Comparisons within each group were made with either a paired Student's t test or repeated-measures ANOVA with post hoc comparisons of changes determined, a priori, to be of interest.

	Results

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Protocol 1: Comparison of ²⁰¹Tl and ^99mTc-N-NOET During Sustained LAD Low Flow
Hemodynamics. Table 1 summarizes the hemodynamic parameters that remained stable throughout the experiment. LAD flow decreased significantly after the setting of the stenosis and remained constant throughout the rest of the experiment. LAD regional wall thickening fell markedly after the stenosis was set and remained akinetic or slightly dyskinetic for the remainder of the experiment. There was no significant difference in LAD systolic thickening at the time points when ²⁰¹Tl and ^99mTc-N-NOET were injected.

View this table: [in this window] [in a new window]   Table 1. Hemodynamic Variables in 10 Dogs With Sustained LAD Low Flow (Protocol 1)

Risk area and infarct size. The LAD risk area by monastral blue dye was 27.9±1.7% of the total left ventricle (range, 20.6% to 36.6%). By TTC staining, only two dogs had small subendocardial myocardial infarction involving 1.6% and 0.8% of the left ventricle, respectively.

Microsphere blood flow and ²⁰¹Tl and^99mTc-N-NOET activities. The mean stenotic-to-normal blood flow ratio was 0.98±0.02 during baseline. It decreased significantly after the stenosis was set and was 0.56±0.07 at the time of ²⁰¹Tl injection (P<.001 versus baseline), 0.52±0.06 at the time of ^99mTc-N-NOET injection (P<.001 versus baseline), and 0.49±0.07 at the end of the experiment (P<.001 versus baseline). The stenotic-to-normal blood flow ratio at the time of ²⁰¹Tl injection was not different from that at the time of ^99mTc-N-NOET injection.

Both ²⁰¹Tl and ^99mTc-N-NOET stenotic-to-normal tissue activity ratios after 2 hours of low flow were significantly higher than the flow ratio at the time of injection (²⁰¹Tl, 0.73±0.04; ^99mTc-N-NOET, 0.76±0.06; P<.001 versus injection flow ratio), indicative of rest redistribution (Fig 2). As shown in the figure, there was no significant difference between ²⁰¹Tl and ^99mTc-N-NOET tissue activity ratios. However, in segments with very low flow (flow reduction >80% from baseline), the ²⁰¹Tl tissue activity ratio was significantly higher than that of ^99mTc-N-NOET (Fig 3). In contrast, in segments with moderate flow reduction (20% to 50% of baseline), ²⁰¹Tl tissue activity ratio was lower than that of ^99mTc-N-NOET.

View larger version (16K): [in this window] [in a new window]   Figure 2. Comparison of 201Tl and 99mTc-N-NOET activities in 10 dogs with sustained coronary artery low flow. Bars on left show comparable stenotic-to-normal blood flow ratios during 201Tl and 99mTc-N-NOET injection. Bars on right show comparable 201Tl and 99mTc-N-NOET stenotic-to-normal tissue activity ratios after 2 hours of sustained low flow. 201Tl and 99mTc-N-NOET tissue activity ratios were significantly higher than flow ratios at time of injection, consistent with tracer redistribution. *P<.001 vs respective blood flow ratio at time of tracer injection.

View larger version (39K): [in this window] [in a new window]   Figure 3. Myocardial blood flow ratios at time of tracer injection and myocardial 201Tl and 99mTc-N-NOET tissue activity ratios after 2 hours of sustained low flow (protocol 1). Results are plotted according to average percentage blood flow reduction between baseline and stenosis. At all levels of flow reduction, 201Tl and 99mTc-N-NOET tissue activity ratios were significantly higher than flow ratios at time they were injected. In very-low-flow (>80% flow reduction) region, 201Tl tissue activity ratios were significantly higher than 99mTc-N-NOET activity ratios; the opposite was observed in the case of a moderate flow reduction (<50% from baseline). *P<.001 vs 201Tl and 99mTc-N-NOET; P<.001 vs 99mTc-N-NOET; P<.05 vs 99mTc-N-NOET.

Image defect ratios. The initial ²⁰¹Tl imaging defect count ratio (0.67±0.03) was not significantly different from the ^99mTc-N-NOET defect count ratio on images acquired 15 minutes after injection (0.66±0.03, P=NS). Thus, in this low-flow model, initial ^99mTc-N-NOET myocardial uptake, like ²⁰¹Tl, reflected the flow disparity at the time of injection. There was a significant increase in the ^99mTc-N-NOET imaging defect count ratio over time (0.73±0.02 at 120 minutes after injection, P<.001 versus 15-minute images) (Fig 4, top). As can be seen in Fig 5, there was significantly faster ^99mTc-N-NOET clearance from the normal-flow LCx zone compared with the hypoperfused LAD zone. The myocardial fractional clearance of ^99mTc-N-NOET was 43.4±3.0% in the normal LCx zone and 37.9±3.1% in the hypoperfused LAD zone (P<.01). In addition, the myocardial ^99mTc-N-NOET clearance half-time was 143±18 minutes in the normal-flow LCx zone and 201±29 minutes in the hypoperfused LAD zone (P=.01), indicating delayed clearance from the low-flow zone.

View larger version (100K): [in this window] [in a new window]   Figure 4. In vivo 99mTc-N-NOET images from representative dogs with sustained low flow (protocol 1) and transient coronary artery occlusion followed by reflow (protocol 2). Initial images were acquired 15 minutes after 99mTc-N-NOET injection. Numbers in parentheses are defect count ratios.

View larger version (16K): [in this window] [in a new window]   Figure 5. Time-activity plot of average cpm per pixel in LAD and LCx ROIs drawn on 99mTc-N-NOET images acquired during sustained low flow (protocol 1, n=10). Note that defect resolution results from faster clearance of 99mTc-N-NOET from normal than from hypoperfused area.

Protocol 2: Myocardial Kinetics of ^99mTc-N-NOET Injected During 15 Minutes of Total LAD Occlusion Followed by 2 Hours of Reflow
Hemodynamics. Table 2 summarizes the hemodynamic data for protocol 2. LAD flow decreased significantly after occlusion at the time ^99mTc-N-NOET injection returned to baseline after reflow and remained constant throughout the experiment. LAD regional wall thickening decreased dramatically after the setting of the stenosis, with no significant variation over time, even after reperfusion.

View this table: [in this window] [in a new window]   Table 2. Hemodynamic Variables in 6 Dogs With Transient Total LAD Occlusion Followed by 2 Hours of Reperfusion (Protocol 2)

Risk area and infarct size. The LAD risk area by monastral blue dye was 27.9±2.7% of the total left ventricle (range, 22.7% to 38.1%). None of the 6 dogs had myocardial infarction by TTC staining.

^99mTc-N-NOET arterial blood activity. ^99mTc-N-NOET blood activity decreased rapidly after injection and was 34.4±4.0%, 13.9±1.7%, 7.1±1.1%, 3.9±0.5%, and 2.7±0.4% of maximal activity at 1, 5, 15, 30, and 120 minutes after injection, respectively.

Microsphere blood flow and ^99mTc-N-NOET tissue activity. The mean LAD-to-LCx blood flow ratio was 0.99±0.03 at baseline, and it decreased to 0.27±0.04 at the time of ^99mTc-N-NOET injection during LAD occlusion (P<.001 versus baseline). After reperfusion, the LAD-to-LCx blood flow ratio increased to 1.19±0.03 (P<.001 versus occlusion) (Fig 6). This ratio was higher than at baseline (P<.01), consistent with a mild transient hyperemic response in the previously occluded region, although LAD flow measured by ultrasonic flow probe at this time had returned to baseline. This hyperemic response resolved over time, and the LAD-to-LCx blood flow ratio at the end of the experiment was slightly lower than at baseline (0.84±0.03, P=.054). The ^99mTc-N-NOET LAD-to-LCx tissue activity ratio after 2 hours of reperfusion (0.84±0.03) was significantly higher than the flow ratio at the time of injection (P<.001) and was identical to the blood flow ratio at the end of the experiment, indicative of complete redistribution of ^99mTc-N-NOET after 2 hours of reflow (Fig 6).

View larger version (22K): [in this window] [in a new window]   Figure 6. Comparison of mean 99mTc-N-NOET tissue activities and myocardial blood flow in 6 dogs with transient coronary artery occlusion (protocol 2). 99mTc-N-NOET LAD-to-LCx tissue activity ratio (hatched bar) was significantly higher than flow ratio at time of injection (solid bar) and was comparable to flow ratio at end of experiment (open bar), consistent with complete 99mTc-N-NOET redistribution over 2 hours.

Image defect ratios. The initial ^99mTc-N-NOET imaging defect count ratio (0.57±0.04) partially reflected the flow disparity at the time of injection but was significantly higher than the LAD-to-LCx flow ratio at the time of injection (0.27±0.04, P<.001), leading to an underestimation of flow disparity. There was a significant increase in the ^99mTc-N-NOET imaging defect ratio over time (P<.001): 0.68±0.04, 0.70±0.04, 0.71±0.04, 0.74±0.04, and 0.75±0.04, respectively, at 15, 30, 60, 90, and 120 minutes after reperfusion (Fig 4, bottom). As shown in Fig 7, there was significantly greater clearance of ^99mTc-N-NOET from the normal-flow LCx area (64±5%) than from the transiently occluded LAD area (52±7%, P<.05). The clearance half-time values for the reflow period were 121±17 and 145±21 minutes for the normal and transiently occluded–reperfused zones, respectively (P<.05).

View larger version (17K): [in this window] [in a new window]   Figure 7. Time-activity plot of average cpm per pixel in LAD and LCx ROIs on in vivo 99mTc-N-NOET images acquired during LAD occlusion (Occ) and 15, 30, 60, 90, and 120 minutes after reflow (protocol 2). Defect resolution resulted from greater clearance of 99mTc-N-NOET from normal LCx area than from transiently occluded–reperfused LAD area.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
^99mTc-N-NOET is the most promising member of the new class of bis-(dithiocarbamato)-nitrido, agents being investigated for myocardial perfusion imaging. ^99mTc-N-NOET has been shown to have a relatively high heart uptake in rats (4.28±0.18% of injected dose), and high-quality myocardial scans have been obtained in preliminary animal studies.¹⁰ The precise mechanism underlying myocardial sequestration and retention of ^99mTc-N-NOET in myocardium remains unclear. Ucelli et al¹¹ showed that in rat myocardium, most of the ^99mTc-N-NOET activity was associated with the hydrophobic components of the cell. No evidence of association of ^99mTc-N-NOET with the cytosol or mitochondria was observed, and the authors concluded that cell membranes are the most likely site of localization of ^99mTc-N-NOET in heart tissue. This concept was confirmed by the study of Johnson et al,²⁰ showing an extremely high retention of ^99mTc-N-NOET in isolated, perfused rat hearts but a markedly increased ^99mTc-N-NOET clearance after cell membrane disruption by Triton X-100.

In a canine model of 50% flow reduction, Ghezzi et al¹² found a high first-pass ^99mTc-N-NOET extraction fraction of 75.5±4% under basal conditions and a linear correlation between myocardial ^99mTc-N-NOET tissue activity and microsphere blood flow 15 minutes after injection (r=.94) . In another group of dogs, these same investigators found that when ^99mTc-N-NOET was injected during low flow followed by 90 minutes of reperfusion, relative tracer activity appeared to increase in the previously ischemic region, consistent with tracer redistribution.¹² Although ^99mTc-N-NOET redistribution can be inferred from these studies, serial imaging was not performed, and thus the precise mechanism of redistribution was unknown. A clinical study comparing stress-redistribution-reinjection ²⁰¹Tl SPECT with stress–delayed rest ^99mTc-N-NOET SPECT in 25 patients determined that the tracers were comparable for detecting the presence and extent of coronary artery disease.¹³ Furthermore, some patients showed normalization of myocardial ^99mTc-N-NOET activity 4 hours after injection, consistent with ^99mTc-N-NOET redistribution. Nevertheless, before this study, ^99mTc-N-NOET had not yet been simultaneously compared with ²⁰¹Tl in controlled experimental models, and the redistribution kinetics of ^99mTc-N-NOET had not been thoroughly assessed.

Data from the present study show ^99mTc-N-NOET redistribution in canine models of sustained low flow and transient coronary artery occlusion followed by reflow. Furthermore, ⁹⁹Tc-N-NOET and ²⁰¹Tl uptake and kinetics are comparable during sustained low flow. The sustained-low-flow model was chosen because it is the model most frequently used to assess the ability of a tracer to redistribute (eg, ²⁰¹Tl) during prolonged hypoperfusion and consequent asynergy when flow is unchanged from the time of tracer administration to the end of the redistribution period. The ability of ^99mTc-NOET imaging to reflect myocardial blood flow disparity and to redistribute over time in the absence of significant myocardial infarction has direct clinical application in the setting of assessing myocardial ischemia with exercise or pharmacological stress. The model of transient occlusion followed by reflow, although less clinically relevant, was performed to better understand the mechanism by which ^99mTc-N-NOET redistributes because it optimizes the conditions for redistribution to occur, ie, transient flow disparity with elevated blood levels of the tracer, and it was the model originally used to define ²⁰¹Tl redistribution kinetics.¹⁴

Sustained Coronary Artery Low Flow
When injected during sustained coronary artery low flow, initial ²⁰¹Tl and ^99mTc-N-NOET defects on initial images were similar and reflected the transmural myocardial blood flow disparity at the time of injection. Serial ^99mTc-N-NOET images showed significant defect resolution over time, which cannot be attributed to resolution of the partial-volume effect in the hypoperfused region, because LAD zone regional systolic wall thickening remained akinetic or dyskinetic throughout the experiment. This redistribution over time at rest resulted from a slower clearance of ^99mTc-N-NOET in the hypoperfused than in the normally perfused regions, rather than accumulation of tracer in the low-flow zone. Postmortem in vitro analysis of myocardial activity confirmed ²⁰¹Tl and ^99mTc-N-NOET redistribution. By gamma-well counting, the ^99mTc-N-NOET stenotic-to-normal tissue activity ratio after 2 hours of low flow was comparable to that of ²⁰¹Tl. For both tracers, this ratio was significantly greater than the stenotic-to-normal blood flow ratio at the time they were injected. Thus, ²⁰¹Tl and ^99mTc-N-NOET kinetics are comparable in this model of sustained low flow. These data are consistent with those of Pohost et al,²¹ who demonstrated that ²⁰¹Tl defects observed soon after tracer injection in dogs with a severe coronary stenosis showed delayed defect resolution over 2 hours. These investigators found that ²⁰¹Tl redistribution was the consequence of both washout of ²⁰¹Tl from the nonischemic regions and accumulation of ²⁰¹Tl in the ischemic myocardium. We observed that ^99mTc-N-NOET defect resolution in this model results predominantly from differential washout, because ^99mTc-N-NOET cleared more rapidly from the normal than the hypoperfused areas and no accumulation of ^99mTc-N-NOET in the low-flow zone was observed. Our model did not permit the concurrent study of ²⁰¹Tl kinetics over time, and we were therefore unable to confirm relative accumulation of ²⁰¹Tl in the hypoperfused zone, as has been previously demonstrated.²¹ These ^99mTc-N-NOET data are in contrast to observations made regarding the degree of delayed rest redistribution of other ^99mTc-labeled perfusion agents. Under conditions of sustained low flow, minimal to no redistribution has been reported for ^99mTc-sestamibi and ^99mTc-tetrofosmin.^{16 22}

Transient Total LAD Occlusion Followed by 2 Hours of Reflow
The initial ^99mTc-N-NOET defect ratio on images acquired 15 minutes after tracer injection during LAD occlusion underestimated the myocardial blood flow disparity at the time of tracer injection. This underestimation of flow discrepancy may have resulted from different but not mutually exclusive mechanisms. ^99mTc-N-NOET uptake in a low-flow area may be higher than the microsphere-determined regional blood flow because of increased extraction fraction of the tracer at low flow rates, as has been demonstrated with ^99mTc-sestamibi or ²⁰¹Tl.^{23 24 25} Another mechanism is that very early redistribution of ^99mTc-N-NOET occurred during the first 15 minutes after injection. Such a rapid tracer clearance from normal myocardium has been shown to occur with ^99mTc-teboroxime, another neutral, highly lipophilic tracer.^{26 27}

In this model, the ^99mTc-N-NOET imaging defect also resolved over time, because of greater clearance of the tracer in the normal region than in the transiently occluded–reperfused region. Interestingly, a significant amount of redistribution occurred during the first 15 to 30 minutes after reflow, suggesting a rapid redistribution of ^99mTc-N-NOET in this model. This early redistribution, although slower than that described for ^99mTc-teboroxime,^{26 27} could be a potential problem for the clinical use of ^99mTc-N-NOET, because some defects might partially resolve before image acquisition. However, in a preliminary study, the sensitivity of ^99mTc-N-NOET scans acquired 30 minutes after injection for the detection of coronary artery disease in patients was not different from that of ²⁰¹Tl.¹³ By gamma-well counting, the ^99mTc-N-NOET LAD-to-LCx myocardial tissue activity ratio after 2 hours of reflow was significantly higher than the LAD-to-LCx blood flow ratio at the time of injection, confirming significant redistribution in this model.

The mechanism of ²⁰¹Tl redistribution in the same transient occlusion and reflow model was investigated in two previous experimental animal studies. Pohost et al¹⁴ demonstrated that when ²⁰¹Tl was injected during transient coronary artery occlusion, initial myocardial ²⁰¹Tl uptake was markedly decreased. After restoration of flow, myocardial ²⁰¹Tl concentration increased over time. Similarly, Beller et al²⁸ established that ²⁰¹Tl activity measured after 10 minutes of occlusion was reduced by 80% compared with normal activity. After reflow, near normalization of ²⁰¹Tl activity between ischemic and nonischemic regions occurred within 4 hours, related to both delayed accumulation of tracer in the previously occluded area and rapid washout from the normal area, as already demonstrated in low-flow models.

In summary, (1) during sustained coronary low flow and transient coronary artery occlusion in open-chest dogs, initial ^99mTc-N-NOET defects on quantitative planar scintigraphy reflect the myocardial blood flow disparity at the time of tracer injection. (2) In both models, ^99mTc-N-NOET undergoes significant redistribution over time. With sustained low flow, ^99mTc-N-NOET undergoes redistribution despite the presence of a 50% flow heterogeneity; however, redistribution in this model was not complete over 2 hours. Interestingly, in this model, ^99mTc-N-NOET and ²⁰¹Tl redistribution are identical, although the two tracers have very different chemical properties. (3) In the transient occlusion model, despite a nearly 75% flow heterogeneity at the time of injection, ^99mTc-N-NOET undergoes complete redistribution, as evidenced by similar blood flow and ^99mTc-N-NOET tissue activity ratios 2 hours after tracer injection. (4) The mechanism of ^99mTc-N-NOET redistribution on serial myocardial scans results from faster clearance in the normal than in the ischemic area.

Study Limitations
^99mTc-N-NOET redistribution kinetics in the present study were assessed only by serial imaging, which is subject to technical difficulties such as scatter, attenuation, partial-volume and motion artifacts, and tissue cross talk. Alternative methods to study tracer redistribution, however, also have significant limitations: Gamma-well counting assessment of tracer kinetics requires killing different groups of animals at different times after injection, and comparison between groups may be altered by subtle changes in experimental conditions such as the degree of ischemia or the hemodynamic parameters. Serial myocardial biopsies can lead to myocardial injury, necessitate the analysis of very small myocardial pieces, and are subject to sampling error. Analysis of regional tracer activity by miniature cadmium telluride probes may be influenced by the changes in myocardial thickness or by activity in the underlying blood pool. A second limitation of this study is the relatively small sample size used in protocol 1 (n=10). This could result in a limited statistical power to detect moderate differences between ²⁰¹Tl and ^99mTc-N-NOET tissue activity ratios. However, such small differences would not likely have important clinical implications with regard to their use for the detection of myocardial ischemia. The transient occlusion model was chosen to optimize the redistribution of ^99mTc-N-NOET and to assess the mechanisms of ^99mTc-N-NOET redistribution on serial images. The clinical implication of this model is limited, however, and the results obtained after a 20-minute occlusion might not apply to a clinical setting of coronary occlusion such as acute myocardial infarction.

Summary
^99mTc-N-NOET is the first ⁹⁹Tc-labeled myocardial perfusion imaging agent with kinetics similar to that of ²⁰¹Tl in experimental canine models, permitting redistribution imaging. Because of better radiophysical properties of ^99mTc, ^99mTc-N-NOET is a very promising agent for the detection and assessment of coronary artery disease.

	Selected Abbreviations and Acronyms

 

LAD = left anterior descending coronary artery LCx = left circumflex coronary artery ROI = region of interest SPECT = single-photon emission computed tomography 99mTc-N-NOET = bis-(N-ethoxy,N-ethyl dithiocarbamato)nitrido 99mTc TTC = triphenyl tetrazolium chloride

	Acknowledgments

 
This work was partially supported by a research grant from CIS-Bio International. Dr Vanzetto was supported by a grant from Parke Davis, France, and Merck Clevenot, France.

	Footnotes

 
Reprint requests to David K. Glover, ME, Cardiovascular Division, Department of Medicine, Box 158, University of Virginia Health Sciences Center, Charlottesville, VA 22908.

Received October 17, 1996; revision received April 28, 1997; accepted May 1, 1997.

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