Evaluation of [(11)C]methyl-losartan and [(11)C]methyl-EXP3174 for PET imaging of renal AT1receptor in rats.

INTRODUCTION: The angiotensin II type 1 receptor (AT1R) is responsible for the main effects of the renin-angiotensin system (RAS), and its expression pattern is altered in several diseases. The [(11)C]methylated derivatives of the clinically used AT1R blocker (ARB) losartan and its active metabolite EXP3174, that binds with higher affinity to AT1R, were evaluated as potential PET imaging tracers in rat kidneys. METHODS: [(11)C]Methyl-losartan and [(11)C]methyl-EXP3174 were synthesized by [(11)C]methylation of the tetrazole-protected analogs using [11C]methyl iodide. Tissue uptake and binding selectivity of [(11)C]methyl-losartan were assessed by ex-vivo biodistribution and in-vitro autoradiography. Radiolabeled metabolites in rat plasma and kidneys were analysed by column-switch HPLC. Both tracers were evaluated with small animal PET imaging. Due to better pharmacokinetics, [(11)C]methyl-EXP3174 was further investigated via PET by co-injection with AT1R antagonist candesartan or the AT2R antagonist PD123,319. RESULTS: Binding selectivity to renal AT1 over AT2 and Mas receptors was demonstrated for [(11)C]methyl-losartan. Plasma metabolite analysis at 10 min revealed stability of [(11)C]methyl-losartan and [(11)C]methyl-EXP3174 with the presence of unchanged tracer at 70.8 ± 9.9% and 81.4 ± 6.0%, of total radioactivity, respectively. Contrary to [(11)C]methyl-losartan, co-injection of candesartan with [(11)C]methyl-EXP3174 reduced the proportion of unchanged tracer (but not metabolites), indicating that these metabolites do not bind to AT1R in rat kidneys. MicroPET images for both radiotracers displayed high kidney-to-background contrast. Candesartan significantly reduced [(11)C]methyl-EXP3174 uptake in the kidney, whereas no difference was observed following PD123,319 indicating binding selectivity for AT1R. CONCLUSIONS: [(11)C]Methyl-EXP3174 displayed a favorable binding profile compared to [(11)C]methyl-losartan for imaging renal AT1Rs supporting further studies to assess its full potential as a quantitative probe for AT1R via PET.

Analysis of [11C]methyl-candesartan kinetics in the rat kidney for the assessment of angiotensin II type 1 receptor density in vivo with PET.

INTRODUCTION: Angiotensin II type 1 (AT(1)) receptors play a key role in the regulation of renal and cardiovascular functions and have been implicated in the pathogenesis of many diseases. The aim of this study was to assess binding of the novel radioligand [(11)C]methyl-candesartan to AT(1) receptors in the rat kidney in vivo with PET. METHODS: Dynamic PET images were acquired for 60 min at baseline, with coinjection of candesartan (5 mg/kg), and after injection of PD123,319 (5 mg/kg). Volumes of distribution (R(C)∙V(T)) were estimated with a two-compartment model, by Logan analysis, and by taking the tissue-to-plasma activity ratio at 50-60 min post-injection. RESULTS: The two-compartment model did not describe the kinetics at baseline adequately and the baseline scans were too short to obtain accurate estimates of R(C)∙V(T) with the Logan approach. Based on the tissue-to-plasma ratios, roughly one-third of V(T) at baseline could be attributed to AT(1) receptor binding. There were no indications of AT(2) receptor binding in the rat kidney. CONCLUSION: It may be possible to detect changes in AT(1) receptor density in the rat kidney in vivo with [(11)C]methyl-candesartan and PET. Imaging AT(1) receptors with PET may provide valuable information on the role of these receptors in the pathogenesis of diseases such as hypertension, diabetic nephropathy, ventricular remodeling, and heart failure.

Uniformity and repeatability of normal resting myocardial blood flow in rats using [13N]-ammonia and small animal PET.

OBJECTIVE: This study aimed to quantitatively evaluate population variability, regional uniformity and repeatability of myocardial blood flow measurements using [13N]-ammonia and small animal PET. METHODS: Serial PET scans were conducted on Sprague-Dawley rats using [13N]-ammonia to study relative perfusion and absolute myocardial blood flow (ml/min/g). FlowQuant automated analysis software was used to produce five-segment polar maps to investigate regional myocardial blood flow differences. The interobserver and intraobserver repeatability was assessed quantitatively using Bland-Altman analysis. RESULTS: Absolute myocardial blood flow values were 4.3 ± 1.1 ml/min/g, corresponding to a population variability of 25.5%. There were significant age-related increases in resting myocardial blood flow (r2=0.59, P<0.001). The test-retest differences had a coefficient of repeatability of 24.5% of the mean myocardial blood flow. The operator variability was small, relative to the population variability. CONCLUSION: Repeatable myocardial blood flow values are minimally influenced by operator intervention. However, age-related myocardial blood flow increases must be taken into account when comparing measurements between experimental groups.

Analysis of (R)- and (S)-[(11)C]rolipram kinetics in canine myocardium for the evaluation of phosphodiesterase-4 with PET.

PURPOSE: (R)-[(11)C]rolipram and (S)-[(11)C]rolipram have been proposed to investigate phosphodiesterase-4 and, indirectly, cAMP-mediated signaling with PET. This study assessed binding of these tracers to phosphodiesterase-4 in canine myocardium. PROCEDURES: Seven dogs underwent (R)-[(11)C]rolipram and (S)-[(11)C]rolipram dynamic PET imaging at baseline and with co-injection of saturating doses of (R)-rolipram. Dual-input compartment models were applied to estimate the volumes of distribution (V(T)). RESULTS: The model comprising one compartment for unmetabolized tracer and one compartment for labeled metabolites provided excellent fits to data acquired with (S)-[(11)C]rolipram at baseline and with both enantiomers during co-injection scans. Use of two compartments for unmetabolized (R)-[(11)C]rolipram at baseline was warranted according to Akaike and Schwarz criteria. V(T) estimates obtained with these models were robust (CV ≤ 8.2%) and reproducible (CV ≤ 15%). CONCLUSION: An important fraction (~65%) of the V (T) of (R)-[(11)C]rolipram at baseline reflects specific binding. Thus, the latter may be a useful index of phosphodiesterase-4 levels in canine myocardium.

PET measurements of cAMP-mediated phosphodiesterase-4 with (R)-[11C]rolipram.

Cyclic adenosine monophosphate (cAMP) is the common second messenger in signal-transduction cascades originating at a number of monoamine receptors involved in neurotransmission, cardiac function and smooth muscle contraction. Altered regulation of cAMP synthesis (at receptors, G-protein subunits or adenylyl cyclase) and breakdown by phosphodiesterase (PDE) enzymes have been implicated in a number of pathologies. The PDE4 inhibitor (R)-rolipram, and the less active (S)- enantiomer, have been labeled with carbon-11 and characterized by in vivo and in vitro experiments for use in the evaluation of altered PDE4 levels in the brain and cardiac tissues. (R)-[11C]Rolipram has been shown to bind selectively to PDE4 over other PDE isozymes, with specific binding reflecting approximately 80 and 40% of the total detected radioactivity in the rat brain and the heart, respectively. Tracer retention in PDE4-rich tissues is increased by cAMP-elevating treatments, as detected by in vivo PET studies and ex vivo biodistribution experiments. In vivo PET imaging studies display strong region-specific signal in the brain and heart, as evaluated in rats, pigs, monkeys and humans. Impaired cAMP-mediated signaling was observed in animal models of aging, obesity, anthracycline-induced cardiotoxicity and myocardial infarction using (R)-[11C]rolipram. Given the critical role of cAMP in multiple hormonal pathways, the good safety profile and well-characterized pharmacokinetics, (R)-[11C]rolipram PET imaging provides a novel tool for serial monitoring of cAMP-mediated signaling at the PDE4 level, yielding insight into pathological progression with potential for directing therapy.

PET of (R)-11C-rolipram binding to phosphodiesterase-4 is reproducible and sensitive to increased norepinephrine in the rat heart.

UNLABELLED: Phosphodiesterase-4 (PDE4) plays a critical role in the regulation of ß-adrenergic receptor-stimulated cyclic adenosine monophosphate cell signaling in the heart. (R)-rolipram, a PDE4-selective inhibitor, has been studied previously as a radiotracer for the quantification of PDE4 levels. The aim of this study was to characterize (R)-(11)C-rolipram binding in the rat myocardium in vivo, using small-animal PET. METHODS: Male Sprague-Dawley rats (n = 30) were administered (R)-(11)C-rolipram and imaged for 60 min to evaluate tracer binding and reproducibility, quantified using Logan slope analysis of the distribution volume. Dynamic (13)N-ammonia imaging was performed to quantify myocardial blood flow and assist in cardiac regional analysis. Saturation studies evaluated the sensitivity of (R)-(11)C-rolipram to PDE4 blocking by unlabeled cold (R)-rolipram (0.0001-1.0 mg/kg), for estimation of the median effective dose (ED(50)) in the heart. (R)-(11)C-rolipram response to enhanced norepinephrine stimulation of the ß-adrenergic receptor with desipramine (20 mg/kg, intravenous) was also studied. Intrarat variability studies (n = 5) were conducted with test-retest imaging at 16 ± 7 d. RESULTS: A reduction of Logan slope was observed with increasing cold mass coadministered with the tracer, with an ED(50) of 0.0019 mg/kg (95% confidence interval, 0.0014-0.0052) estimated from the saturation studies. This ED(50) predicted less than 10% enzyme occupancy at 0.0002 mg of cold (R)-rolipram per kilogram (mass/body weight). Low-occupancy imaging at 0.00018 ± 0.00002 mg/kg produced a mean Logan slope of 5.5 ± 0.85 mL/cm(3). Enzyme saturation of more than 90%, compared with low-occupancy conditions, occurred at more than 0.02 mg/kg, with a complete blocking dose (>1 mg of (R)-rolipram per kilogram) resulting in a Logan slope of 3.3 ± 0.1 mL/cm(3), representing a 40% reduction. Compared with baseline, a Logan slope of 6.8 ± 0.7 mL/cm(3) in desipramine-challenged animals was observed, representing a 30% increase due to acute norepinephrine stimulation, despite a reduction in myocardial blood flow. Intrarat and intraoperator variability was less than 5% between repeated measures. CONCLUSION: (R)-(11)C-rolipram shows the ability to monitor increases and decreases in PDE4 availability in the rat myocardium, with good reproducibility.

3D versus 2D dynamic 82Rb myocardial blood flow imaging in a canine model of stunned and infarcted myocardium.

PURPOSE: Previous studies have shown the ability of rubidium-82 ((82)Rb) positron emission tomography (PET) imaging to quantitatively measure myocardial blood flow (MBF), many of which are performed using two-dimensional (2D) imaging. Three-dimensional (3D) imaging provides increased sensitivity and may result in decreased costs owing to a reduction in the required injected activity of radiotracer. This study compares 2D and 3D (82)Rb PET MBF results obtained in the same imaging session. METHODS: Three-dimensional and 2D (82)Rb perfusion imaging was performed in canines on a GE Discovery LS PET/CT scanner at rest and during hyperemia in stunned and infarcted tissue. MBF (ml/min/g) was determined using a 1-compartment model and an extraction correction of the uptake rate and analyzed using a standard 17-segment model. RESULTS: A strong, significant correlation was present (rho = 0.95, P<0.0001). Average 3D MBF values were slightly lower at rest and higher during stress versus 2D. MBF results in normal, stunned, and infarcted tissue differed by 7% on average and significant increases in MBF from rest to hyperemia were noted with both the techniques. CONCLUSION: These results imply that MBF results obtained in 3D are comparable with traditional 2D imaging. Therefore, it may be possible to use 3D imaging with lower administered activity, helping to reduce costs and patient dose without compromising quantitative information.

Quantification of regional myocardial blood flow in a canine model of stunned and infarcted myocardium: comparison of rubidium-82 positron emission tomography with microspheres.

BACKGROUND: Myocardial viability and quantification of regional myocardial blood flow (MBF) are important for the diagnosis of heart disease. Positron emission tomography is the current gold standard for determining myocardial viability, but most positron-emitting perfusion tracers require an on-site cyclotron. Rubidium-82 ((82)Rb) is a myocardial perfusion tracer that is produced using an on-site generator. This study investigates (82)Rb-measured MBF in canine models of stunned and infarcted myocardium compared with selected measurements obtained concurrently using microspheres. METHODS: Myocardial stunning and infarction were created in canines by occluding the left anterior descending for 15 min and 2 h, respectively. Stunning was produced in all animals; six animals were reperfused after the 2 h occlusion, whereas the other six animals remained occluded permanently. Regional MBF was measured in each group during rest and dobutamine stress at acute and chronic (8 weeks postinsult) time points using dynamic (82)Rb perfusion imaging and radioactively labeled microspheres. RESULTS: Average resting MBF with microspheres and Rb was 0.68+/-0.02 versus 0.73+/-0.01 (P<0.001) in nonischemic tissue, and 0.53+/-0.03 versus 0.42+/-0.02 (P<0.001) in the region-at-risk tissue, respectively. Average MBF during stress with microspheres and Rb was 2.78+/-0.15 versus 3.53+/-0.16 (P<0.05) in the nonischemic tissue, and 1.90+/-0.20 versus 2.31+/-0.26 (P = NS) in the region-at-risk tissue, respectively. CONCLUSION: Despite the small significant differences, the dynamic (82)Rb measurements provide estimates of MBF in stunned and acutely and chronically infarcted tissue at rest and during hyperemia that correspond with clinical interpretation.

Use of a column-switching high-performance liquid chromatography method to assess the presence of specific binding of (R)- and (S)-[(11)C]rolipram and their labeled metabolites to the phosphodiesterase-4 enzyme in rat plasma and tissues.

INTRODUCTION: To complement recent studies using the high-affinity (11)C-labeled phosphodiesterase-4 (PDE4) inhibitor (R)-rolipram and the less active enantiomer (S)-[(11)C]rolipram for in vivo quantification of PDE4 levels, we evaluated the presence of radiolabeled metabolites and their potential binding to PDE4 in the rat plasma, brain, heart, pancreas, skeletal muscle and brown adipose tissue. METHODS: A reverse-phase capture and analytical HPLC column-switch method was used to detect (R)-[(11)C]rolipram, (S)-[(11)C]rolipram and their radiolabeled metabolites in rat plasma and tissue extracts. The relative proportion of PDE4-specific binding of the radiotracers and their labeled metabolites was analyzed following co-injections with a saturating dose of unlabeled (R)-rolipram at 45 min post-tracer injection in tissue extracts. RESULTS: Radiolabeled metabolites were found in the plasma (72-75% of total radioactive signal), and in the heart, skeletal muscle, pancreas and brown adipose tissue (44-52%), but not in the brain. In comparison to polar labeled metabolites, the proportion of unchanged (R)-[(11)C]rolipram was reduced in PDE4-rich organs by co-injection of unlabeled (R)-rolipram. Conversely, no changes were obtained in brown adipose tissue, or with (S)-[(11)C]rolipram, suggesting that radiolabeled metabolites of (R)-[(11)C]rolipram display no specific binding to PDE4. CONCLUSIONS: Radiolabeled hydrophilic metabolites are unlikely to compete with (R)-[(11)C]rolipram for PDE4-specific retention. However, due to the high proportion of the radioactive metabolites in the total radioactive signal, any kinetic modeling calculations in the peripheral tissues will need to take into account the presence of labeled metabolites.

Concomitant treatment with oral L-arginine improves the efficacy of surgical angiogenesis in patients with severe diffuse coronary artery disease: the Endothelial Modulation in Angiogenic Therapy randomized controlled trial.

OBJECTIVE: Endothelial dysfunction and decreased nitric oxide bioavailability may explain why therapeutic angiogenesis and cell therapy have mostly failed in humans. Building from previous large animal work, the Phase I Endothelial Modulation in Angiogenic Therapy trial tested the hypothesis that L-arginine, a nitric oxide donor, may be safe and effective in potentiating surgical angiogenesis in humans. METHODS: Patients with surgical triple-vessel coronary disease and a severely diffusely diseased left anterior descending artery were randomized in 2 x 2 factorial fashion to receive ten 200-microg injections of vascular endothelial growth factor-165 plasmid DNA or placebo in the anterior myocardium along the proximal and mid-left anterior descending arteries, plus oral L-arginine supplementation at a dose of 6 g per day or placebo for 3 months. The distal left anterior descending artery and other coronary arteries were grafted. End points included 3-month changes in myocardial perfusion and contractility of the anterior myocardium, using (13)N-ammonia positron emission tomography and echocardiography. Baseline scans were obtained 3 to 7 days postoperatively to delineate treatment effects from the effects of coronary artery bypass grafting. RESULTS: Patient (N = 19) characteristics were equivalent between groups. There was no perioperative or late mortality. Patients who received the combination of vascular endothelial growth factor and L-arginine had improved anterior wall perfusion on positron emission tomography (P = .02), a trend toward smaller perfusion defects (P = .10), and better anterior wall contractility (P = .02, Kruskal-Wallis) at 3 months versus baseline. This was corroborated by a trend toward better disease perception at 3 months versus baseline on the Seattle Angina Questionnaire (score improvement of 47 +/- 35, combination treatment group; P = .1, Kruskal-Wallis). CONCLUSION: To our knowledge, this is the first study to examine concomitant substrate modification in patients undergoing new biosurgical therapies by using vascular endothelial growth factor angiogenesis. The results suggest safety and efficacy. Concomitant endothelial modulation with L-arginine not only has the potential to make angiogenesis effective but also may have implications for cell therapy trials.

Quantification of myocardial blood flow with 82Rb dynamic PET imaging.

PURPOSE: The PET tracer (82)Rb is commonly used to evaluate regional perfusion defects for the diagnosis of coronary artery disease. There is limited information on the quantification of myocardial blood flow and flow reserve with this tracer. The goal of this study was to investigate the use of a one-compartment model of (82)Rb kinetics for the quantification of myocardial blood flow. METHODS: Fourteen healthy volunteers underwent rest and dipyridamole stress imaging with both (13)N-ammonia and (82)Rb within a 2-week interval. Myocardial blood flow was estimated from the time-activity curves measured with (13)N-ammonia using a standard two-compartment model. The uptake parameter of the one-compartment model was estimated from the time-activity curves measured with (82)Rb. To describe the relationship between myocardial blood flow and the uptake parameter, a nonlinear extraction function was fitted to the data. This function was then used to convert estimates of the uptake parameter to flow estimates. The extraction function was validated with an independent data set obtained from 13 subjects with documented evidence of coronary artery disease (CAD). RESULTS: The one-compartment model described (82)Rb kinetics very well (median R-square = 0.98). The flow estimates obtained with (82)Rb were well correlated with those obtained with (13)N-ammonia (r = 0.85), and the best-fit line did not differ significantly from the identity line. Data obtained from the subjects with CAD confirmed the validity of the estimated extraction function. CONCLUSION: It is possible to obtain accurate estimates of myocardial blood flow and flow reserve with a one-compartment model of (82)Rb kinetics and a nonlinear extraction function.

In vivo selective binding of (R)-[11C]rolipram to phosphodiesterase-4 provides the basis for studying intracellular cAMP signaling in the myocardium and other peripheral tissues.

INTRODUCTION: Phosphodiesterase-4 (PDE4) enzymes specifically break down the second messenger cAMP, thereby terminating the intracellular signaling cascade that plays an essential role in neurohormonal modulation of many physiological systems. PDE4 activity and expression are regulated by cAMP levels, suggesting that measurement of PDE4 provides an index of intracellular cAMP signaling. METHODS: Male Sprague-Dawley rats were administered (R)- or the less active enantiomer (S)-[11C]rolipram and sacrificed 30 min later with tracer retention measured in various tissues. Co-injections with saturating doses of unlabeled (R)-rolipram, (S)-rolipram and Ro 20-1724, as well as subtype-selective PDE inhibitors vinpocetine, Bay 60-7550, cilostazol and zaprinast were used to establish binding selectivity for PDE4 over PDE1, PDE2, PDE3 and PDE5 subtypes, respectively. Autoradiography was performed to substantiate results of biodistribution studies in the myocardium. RESULTS: In vivo (R)-[11C]rolipram retention was dose-dependently reduced by co-injections of (R)-rolipram and (S)-rolipram (ED50 values of 0.03 mg/kg and 0.2 mg/kg, respectively). Vinpocetine, Bay 60-7550, cilostazol and zaprinast had no effect on (R)-[11C]rolipram binding, while (R)-rolipram and Ro 20-1724 reduced the tracer uptake to nonspecific levels in PDE4-rich tissues. CONCLUSIONS: In addition to the brain, (R)-[11C]rolipram binds selectively to PDE4 across all cardiac regions, skeletal muscle, lungs and pancreas, but not in the adipose tissues. In vivo findings were confirmed by in vitro autoradiography studies, suggesting that (R)-[11C]rolipram can be applied to evaluate alterations in central and peripheral PDE4 levels and cAMP-mediated signaling.

Effect of exercise training on myocardial blood flow in patients with stable coronary artery disease.

BACKGROUND: The mechanisms by which exercise training benefits patients with coronary artery disease (CAD) are unclear but may include improved myocardial circulation. The aim of this study was to investigate the effect of exercise training on myocardial blood flow (MBF) and coronary flow reserve (CFR) in patients with stable CAD. METHODS: Twelve patients with documented CAD and ischemic ST-segment depression during exercise testing were randomized to exercise training (n = 7) or sedentary life style (control; n = 5) and underwent rubidium-82 positron emission tomography pre- and postintervention. Global left ventricle MBF and regional MBF in 17 left ventricular segments were calculated. Segments with <75% uptake (2 SD below normal) on stress uptake images were defined as abnormal. RESULTS: Exercise training increased global CFR by 20.8% +/- 27.9% versus control (10.5 +/- 24.1%, P = .0001). In normal segments (exercise training: n = 91; control: n = 46), exercise training did not change resting MBF (-14.1% +/- 16.3% vs -8.8% +/- 15.6%) and hyperemic MBF (-1.93% +/- 19.1% vs 2.86% +/- 20.5%, P = NS) and increased in CFR compared to control (17.0% +/- 25.5% vs 11.3% +/- 23.5%, P = .01). In abnormal segments, the change in resting MBF was not significantly different (-12.6% +/- 18.5% exercise [28 segments] vs -2.9% +/- 18.0% control [39 segments], P = NS). A significant increase was seen in hyperemic MBF with exercise (12.5% +/- 22.1% vs 2.6% +/- 16.3%, P = .02) and CFR (32.8% +/- 32.3% vs 9.5% +/- 24.8%, P = .001). CONCLUSIONS: Exercise training increased CFR in normal and diseased segments, and increased hyperemic flow in diseased segments. These data provide preliminary evidence in support of a favorable effect of exercise training on blood flow to ischemic myocardium.