Miran Kenk

PET of (R)-11C-Rolipram Binding to Phosphodiesterase-4 Is Reproducible and Sensitive to Increased Norepinephrine in the Rat Heart

Phosphodiesterase-4 (PDE4) plays a critical role in the regulation of β-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)-11C-rolipram binding in the rat myocardium in vivo, using small-animal PET. Methods: Male Sprague–Dawley rats (n = 30) were administered (R)-11C-rolipram and imaged for 60 min to evaluate tracer binding and reproducibility, quantified using Logan slope analysis of the distribution volume. Dynamic 13N-ammonia imaging was performed to quantify myocardial blood flow and assist in cardiac regional analysis. Saturation studies evaluated the sensitivity of (R)-11C-rolipram to PDE4 blocking by unlabeled cold (R)-rolipram (0.0001–1.0 mg/kg), for estimation of the median effective dose (ED50) in the heart. (R)-11C-rolipram response to enhanced norepinephrine stimulation of the β-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 ED50 of 0.0019 mg/kg (95% confidence interval, 0.0014–0.0052) estimated from the saturation studies. This ED50 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/cm3. 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/cm3, representing a 40% reduction. Compared with baseline, a Logan slope of 6.8 ± 0.7 mL/cm3 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)-11C-rolipram shows the ability to monitor increases and decreases in PDE4 availability in the rat myocardium, with good reproducibility.

Use of a column-switching high-performance liquid chromatography method to assess the presence of specific binding of (R)- and (S)-[11C]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.

Reduced CGP12177 binding to cardiac β-adrenoceptors in hyperglycemic high-fat-diet-fed, streptozotocin-induced diabetic rats

INTRODUCTION Abnormal sympathetic nervous system and β-adrenoceptor (β-AR) signaling is associated with diabetes. [(3)H]CGP12177 is a nonselective β-AR antagonist that can be labeled with carbon-11 for positron emission tomography. The aim of this study was to examine the suitability of this tracer for evaluation of altered β-AR expression in diabetic rat hearts. METHODS Ex vivo biodistribution with [(3)H]CGP12177 was carried out in normal Sprague-Dawley rats for evaluation of specific binding and response to continuous β-AR stimulation by isoproterenol. In a separate group, high-fat-diet feeding imparted insulin resistance and a single intraperitoneal injection of streptozotocin (STZ) or vehicle evoked hyperglycemia (blood glucose >11 mM). [(3)H]CGP12177 biodistribution was assessed at 2 and 8 weeks post-STZ to measure β-AR binding in heart, 30 min following tracer injection. Western blotting of β-AR subtypes was completed in parallel. RESULTS Infusion of isoproterenol over 14 days did not affect cardiac binding of [(3)H]CGP12177. Approximately half of rats treated with STZ exhibited sustained hyperglycemia and progressive hypoinsulinemia. Myocardial [(3)H]CGP12177 specific binding was unchanged at 2 weeks post-STZ but significantly reduced by 30%-40% at 8 weeks in hyperglycemic but not euglycemic STZ-treated rats compared with vehicle-treated controls. Western blots supported a significant decrease in β(1)-AR in hyperglycemic rats. CONCLUSIONS Reduced cardiac [(3)H]CGP12177 specific binding in the presence of sustained hyperglycemia corresponds to a decrease in relative β(1)-AR expression. These data indirectly support the use of [(11)C]CGP12177 for assessment of cardiac dysfunction in diabetes.

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.

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

Purpose(R)-[11C]rolipram and (S)-[11C]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.ProceduresSeven dogs underwent (R)-[11C]rolipram and (S)-[11C]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 (VT).ResultsThe model comprising one compartment for unmetabolized tracer and one compartment for labeled metabolites provided excellent fits to data acquired with (S)-[11C]rolipram at baseline and with both enantiomers during co-injection scans. Use of two compartments for unmetabolized (R)-[11C]rolipram at baseline was warranted according to Akaike and Schwarz criteria. VT estimates obtained with these models were robust (CV ≤ 8.2%) and reproducible (CV ≤ 15%).ConclusionAn important fraction (~65%) of the VT of (R)-[11C]rolipram at baseline reflects specific binding. Thus, the latter may be a useful index of phosphodiesterase-4 levels in canine myocardium.