Sensitivity of [11C]phenylephrine kinetics to monoamine oxidase activity in normal human heart.

UNLABELLED: Phenylephrine labeled with 11C was developed as a radiotracer for imaging studies of cardiac sympathetic nerves with PET. A structural analog of norepinephrine, (-)-[11C]phenylephrine (PHEN) is transported into cardiac sympathetic nerve varicosities by the neuronal norepinephrine transporter and stored in vesicles. PHEN is also a substrate for monoamine oxidase (MAO). The goal of this study was to assess the importance of neuronal MAO activity on the kinetics of PHEN in the normal human heart. MAO metabolism of PHEN was inhibited at the tracer level by substituting deuterium atoms for the two hydrogen atoms at the alpha-carbon side chain position to yield the MAO-resistant analog D2-PHEN. METHODS: Paired PET studies of PHEN and D2-PHEN were performed in six normal volunteers. Hemodynamic and electrocardiographic responses were monitored. Blood levels of intact radiotracer and radiolabeled metabolites were measured in venous samples taken during the 60 min dynamic PET study. Myocardial retention of the tracers was regionally quantified as a retention index. Tracer efflux between 6 and 50 min after tracer injection was fit to a single exponential process to obtain a washout half-time for all left ventricular regions. RESULTS: Although initial heart uptake of the two tracers was similar, D2-PHEN cleared from the heart 2.6 times more slowly than PHEN (mean half-time 155+/-52 versus 55+/-10 min, respectively; P < 0.01). Correspondingly, heart retention of D2-PHEN at 40-60 min after tracer injection was higher than PHEN (mean retention indices 0.086+/-0.018 versus 0.066+/-0.011 mL blood/ min/mL tissue, respectively; P < 0.003). CONCLUSION: Efflux of radioactivity from normal human heart after uptake of PHEN is primarily due to metabolism of the tracer by neuronal MAO. Related mechanistic studies in the isolated rat heart indicate that vesicular storage of PHEN protects the tracer from rapid metabolism by neuronal MAO, suggesting that MAO metabolism of PHEN leaking from storage vesicles leads to the gradual loss of PHEN from the neurons. Thus, although MAO metabolism influences the rate of clearance of PHEN from the neurons, MAO metabolism is not the rate-determining step in the observed efflux rate under normal conditions. Rather, the rate at which PHEN leaks from storage vesicles is likely to be the rate-limiting step in the observed efflux rate.

Clinical evaluation of carbon-11-phenylephrine: MAO-sensitive marker of cardiac sympathetic neurons.

UNLABELLED: The sympathomimetic drug phenylephrine recently has been labeled with 11C for use in PET studies of cardiac sympathetic innervation. Previous reports using isolated perfused rat heart models indicate that phenylephrine is metabolized by intraneuronal monoamine oxidase (MAO). This report compares the imaging characteristics, neuronal selectivity and kinetics of (-)-[11C]phenylephrine (PHEN) to the structurally similar but MAO-resistant analog (-)-[11C]-meta-hydroxyephedrine (HED), an established heart neuronal marker. METHODS: Fourteen healthy volunteers were studied with PET and PHEN. Ten had paired studies with HED; four of the 10 were scanned a second time with each tracer after oral administration of desipramine, a selective neuronal transport blocker. Hemodynamic and electrocardiographic responses were monitored. Blood levels of intact radiotracer and radiolabeled metabolites were determined from venous blood samples taken during the PET study. Myocardial retention indices for both tracers were calculated. RESULTS: No hemodynamic or electrocardiographic effects were observed with either tracer. PHEN showed reduced myocardial retention at 50 min compared to HED; however, image quality and uniformity of distribution were comparable. PHEN cleared from myocardium with a mean half-time of 59 +/- 5 min, while myocardial levels of HED remained constant. PHEN metabolites appeared in the blood approximately three times faster than HED metabolites. Desipramine pretreatment markedly reduced (> 60%) myocardial retention of both PHEN and HED. CONCLUSION: PHEN provides PET images of human heart comparable in quality and uniformity to HED. Like HED, PHEN localizes in the sympathetic nerves of the heart. However, the more rapid efflux of PHEN, that is likely mediated by MAO, may provide information on the functional status of cardiac sympathetic neurons unobtainable with HED.

Vesamicol receptor mapping of brain cholinergic neurons with radioiodine-labeled positional isomers of benzovesamicol.

UNLABELLED: Alzheimer's disease is characterized by progressive cerebral cholinergic neuronal degeneration. Radiotracer analogs of benzovesamicol, which bind with high affinity to the vesamicol receptor located on the uptake transporter of acetylcholine storage vesicles, may provide an in vivo marker of cholinergic neuronal integrity. Five positional isomers of racemic iodobenzovesamicol (4'-, 5-, 6-, 7-, and 8-IBVM) were synthesized, exchange-labeled with iodine-125, and evaluated as possible in vivo markers for central cholinergic neurons. Only two isomers, 5-IBVM (5) and 6-IBVM (10), gave distribution patterns in mouse brain consistent with cholinergic innervation: striatum >> hippocampus > or = cortex > hypothalamus >> cerebellum. The 24-h tissue-to-cerebellum concentration ratios for 5-IBVM (5) were 3-4-fold higher for striatum, cortex, and hippocampus than the respective ratios for 6-IBVM (10). Neither 8-IBVM (16) nor 4'-IBVM (17) exhibited selective retention in any of the brain regions examined. In the heart, only 5-IBVM (5) exhibited an atria-to-ventricles concentration ratio consistent with high peripheral cholinergic neuronal selectivity. The 7-IBVM (14) isomer exhibited an anomalous brain distribution pattern, marked by high and prolonged retention in the five brain regions, most notably the cerebellum. This isomer was screened for binding in a series of 26 different biological assays; 7-IBVM (14) exhibited affinity only for the delta-receptor with an IC50 of approximately 30 nM. Drug-blocking studies suggested that brain retention of 7-IBVM (14) reflects high-affinity binding to both vesamicol and delta-receptors. Competitive binding studies using rat cortical homogenates gave IC50 values for binding to the vesamicol receptor of 2.5 nM for 5-IBVM (5), 4.8 nM for 6-IBVM (10), and 3.5 nM for 7-IBVM (14). Ex vivo autoradiography of rat brain after injection of (-)-5-[125I]IBVM ((-)-[125I]5) clearly delineated small cholinergic-rich areas such as basolateral amygdala, interpeduncular nucleus, and facial nuclei. Except for cortex, regional brain levels of (-)-5-[123I]IBVM ((-)-[123I]5) at 4 h exhibited a linear correlation (r2 = 0.99) with endogenous levels of choline acetyltransferase. CONCLUSION: Vesamicol receptor mapping of cholinergic nerve terminals in murine brain can be achieved with 5-IBVM (5) and less robustly with 6-IBVM (10), whereas the brain localization of 7-IBVM (14) reflects high-affinity binding to both vesamicol and delta-receptors.

Synthesis and evaluation of [123I]-iodo-PK11195 for mapping peripheral-type benzodiazepine receptors (omega 3) in heart.

An iodinated analog of PK11195, 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)isoquinoline-3-carboxamide , a specific antagonist of the peripheral-type benzodiazepine receptor (omega 3), has been synthesized in three steps with an overall chemical yield of 40%. Both [123I]- and [125I]-Iodo-PK11195 have been synthesized by solid-state isotopic exchange in > 60% isolated radiochemical yield and specific activity of 233-348 mCi/mmol. Tissue distribution studies in rats indicate a high uptake of radioactivity in adrenal glands, heart, lung and kidneys, which was blocked 63-87% by preadministration of cold PK11195. Single photon emission computer tomography (SPECT) imaging of the canine heart has been accomplished with [123I]PK11195. These results suggest that [123I]PK11195 has potential as a SPECT radiotracer for studying the omega 3 receptor in humans.

Direct optical resolution of vesamicol and a series of benzovesamicol analogues by high-performance liquid chromatography.

The direct optical resolution of the vesicular acetylcholine uptake inhibitors vesamicol and benzovesamicol and nine benzovesamicol analogues were performed by HPLC on a commercially available cellulose tris(3,5-di-methylphenyl carbamate) chiral stationary phase. Separation of each enantiomeric pair was optimized with respect to solvent strength and flow-rate, using mobile phase mixtures of hexane-2-propanol-diethylamine. The method has been successfully applied to the analysis of the optical purity of benzovesamicol intermediates and products, including (-)-5-[123I]iodobenzovesamicol which is currently undergoing clinical evaluation as a tracer for mapping central cholinergic neurons, and the purification of both antipodes of (+/-)-7-[125I]iodobenzovesamicol.

In vivo mapping of cholinergic neurons in the human brain using SPECT and IBVM.

UNLABELLED: In the search for an in vivo marker of cholinergic neuronal integrity, we extended to human use the tracer (-)-5-[123I]iodobenzovesamicol (IBVM). METHODS: IBVM, an analog of vesamicol that binds to the acetylcholine transporter on presynaptic vesicles, was prepared with specific activity greater than 1.11 x 10(9) MBq mmole-1. After intravenous injection of [123I]IBVM, body distribution studies (n = 5) and brain SPECT studies (n = 5) were performed on normal human subjects (n = 10). SPECT images of the brain were collected sequentially over the first 4.5 hr following injection, and again 18 hr later. Data were realigned and transformed to stereotaxic coordinates, and localized activities were extracted for tracer kinetic analysis. The cerebral tracer input function was determined from metabolite-corrected radial arterial blood samples. The best data fit was obtained using a three-compartment model, including terms reflecting cerebral blood volume, exchange of free tracer between plasma and brain and specific binding. RESULTS: Dissociation of bound tracer was negligible for up to 4 hr. For the fitted parameters reflecting transport (K1) and binding site density index (k3), coefficients of variation were approximately 8% in cortical regions of interest. Relative distributions corresponded well with postmortem immunohistochemical values reported for the acetylcholine-synthesizing enzyme choline acetyltransferase, k3 (IBVM binding site density index), and tracer activity distribution at 22 hr, but not at 4 hr after injection. CONCLUSION: SPECT imaging of [123I]IBVM succeeds as an in vivo measure of cholinergic neuronal integrity and should be useful for the study of cerebral degenerative processes such as Alzheimer's disease.

High yield synthesis of high specific activity R-(-)-[11C]epinephrine for routine PET studies in humans.

R-(-)-[11C]Epinephrine ([11C]EPI) has been synthesized from R-(-)-norepinephrine by direct methylation with [11C]methyl iodide or [11C]methyl triflate. The total synthesis time including HPLC purification was 35-40 min. The radiochemical yields (EOB) were 5-10% for [11C]methyl iodide and 15-25% for [11C]methyl triflate. Radiochemical purity was > 98%; optical purity determined by radio-chiral HPLC was > 97%. The [11C]methyl triflate technique produces R-(-)-[11C]epinephrine in quantities (80-170 mCi) sufficient for multiple positron emission tomography studies in humans. The two synthetic methods are generally applicable to the production of other N-[11C]methyl phenolamines and N-[11C]methyl catecholamines.

Synthesis of the 123I- and 125I-labeled cholinergic nerve marker (-)-5-iodobenzovesamicol.

The highly toxic curraremimetic and cholinergic neuron marker (-)-5-iodobenzovesamicol (IBVM) has been labeled with iodine-125 and iodine-123. [125I]IBVM, suitable for animal distribution and ex vivo autoradiographic studies, was synthesized by solid-state exchange; isolated yields were 65-89% with specific activities in the range of 130-200 Ci/mmol. The synthesis of no-carrier-added (-)-5-[125I]IBVM from the corresponding chiral (-)-5-(tri-n-butyltin) derivative using Na125I was evaluated using the oxidants H2O2, peracetic acid and chloramine-T. Both peracetic acid and chloramine-T gave good yields (70-95%). However, when Na123I was utilized, acceptable yields of [123I]IBVM were obtained only with chloramine-T. Use of the latter oxidant did produce 5-chlorobenzovesamicol which was eliminated during HPLC purification. After optimization of the reaction parameters, [123I]IBVM in batch sizes of 10-27 mCi, is routinely obtained with a specific activity of 30-70,000 Ci/mmol, radiochemical purity (> 97%) and chiral purity (> 98%). Isolated radiochemical yields have averaged 71% (N = 40). Distribution analyses of [125I]IBVM and [123I]IBVM in mice 4 h following intravenous administration show essentially equivalent concentrations of the two tracers in the four brain regions sampled. The exceptionally high specific activity of [123I]IBVM has made possible the evaluation of this radiotracer in humans.

Synthesis of a nonpeptide carbon-11 labeled substance P antagonist for PET studies.

CP 96,345 is a nonpeptide high affinity antagonist of the substance P (NK1) receptor. The radiosynthesis of [11C]CP 96,345 suitable for Positron Emission Tomography (PET) applications is described. [11C]CP 96,345 was prepared by O-methylation of a desmethyl precursor via in situ generation of its phenolate salt. The in vivo tissue distribution of [11C]CP 96,345 in guinea pigs (n = 2) at 5 and 30 min was determined. Uptake was low in brain (approximately 0.04% dose/g) and highest (approximately 1-2% dose/g) in the spleen and lungs. The present findings indicate that the use of [11C]CP 96,345 in PET might be more applicable to the study of substance P receptors in peripheral tissues involved with inflammatory disease and arthritis.

Iodine-125 and fluorine-18 labeled aryl-1,4-dialkylpiperazines: potential radiopharmaceuticals for in vivo study of the dopamine uptake system.

A series of fluorine-18 and iodine-125 labeled aryl-1,4-dialkylpiperazine analogs, derivatives of GBR 12935, were synthesized as radiotracers for positron emission tomography or single photon emission computerized tomography imaging of the brain based on their affinity for the presynaptic dopamine reuptake system. High specific activity fluorine-18 tracers were prepared by nucleophilic aromatic substitution reactions; iodine-125 tracers were prepared by isotopic exchange reactions. In vitro competitive binding studies demonstrated that iodine substitution is tolerated in the 4-position of the phenyl ring of the phenalkylpiperazine group. In vivo regional brain biodistribution studies in mice indicated no selectivity of the radioiodinated ligands for the dopamine reuptake site, with striatum/cerebellum concentration ratios of 1. Similar negative results with the new fluorine-18 derivatives demonstrated that in vivo selectivity for the dopamine reuptake site appears to be critically dependent on the carbon chain length between the piperazine ring and the solitary aromatic ring. These studies suggest that development of new radiopharmaceuticals based on the GBR 12935 structure cannot be based solely on considerations of in vitro binding affinities.

Scintigraphic portrayal of beta receptors in the heart.

Myocardial beta adrenergic receptors play important roles in physiology and disease, but the receptors have not before been portrayed. The beta antagonist, iodocyanopindolol (ICYP), was used to develop a scintigraphic method for depicting the receptors in the living heart. Labeled with 125I, ICYP bound firmly to beta receptors in the rat heart; the data conformed to a mathematical model. In vivo saturation kinetics indicated binding sites with two affinities. Inhibition of ICYP binding by beta antagonists of different potency and different selectivity for beta-1 and beta-2 receptors produced the expected pharmacologic effects. Inhibition by lipophilic and hydrophilic antagonists gave no evidence that ICYP was appreciably bound to internalized receptors. Fractional binding by tracer quantities of (-) ICYP and (+/-) ICYP demonstrated stereospecificity. Labeled with 123I, ICYP bound to the hearts of intact dogs so that scintigraphic tomographs depicted ventricular myocardium. Small doses of beta antagonists selectively reduced the binding of ICYP to lung enabling better visualization of the heart. Thus, 123I-ICYP appears to portray the beta receptors in the living heart, and the characteristics of binding permit the development of mathematical models and lay the basis for quantifying this receptor binding.

A rapid high yield synthesis of no-carrier-added (-)-[123I]iodocyanopindolol.

A convenient and efficient radiosynthesis of no-carrier-added [123I]labeled (-)iodocyanopindolol, (-)-[123I]ICYP, a high affinity beta adrenergic antagonist, is described. (-)-[123I]ICYP was synthesized by a modified chloramine-T radioiodination of (-)cyanopindolol followed by a novel reversed-phase HPLC purification that provided the radiopharmaceutical as a directly injectable solution. The total synthesis time was typically less than 45 min and provided (-)-[123I]ICYP in a 59% radiochemical yield (not corrected for decay). In view of its high affinity for the beta adrenergic receptor, (-)-[123I]ICYP is a potentially useful probe for SPECT evaluation of cardiac adrenergic receptor density.

Dual mechanisms of inhibition of DNA synthesis by triciribine.

The inhibition of DNA synthesis in triciribine (TCN)-treated L1210 cells was shown to involve two mechanisms, with different concentration dependence. (a) Initiation of new replicons and possibly of Okazaki fragments was inhibited when the cells were treated with 0.1 microM TCN. The inhibition of replicon initiation was shown by the rate of alkaline elution of [3H]DNA from 15-min-[3H]thymidine-labeled cells being slower if the cells had been pretreated with TCN, indicating that the average size of actively replicating DNA strands was increased. (b) At 1 microM TCN elongation of previously initiated DNA chains was also inhibited. This conclusion was suggested by the decrease in the rate of alkaline elution of [3H]DNA, during postlabeling incubation, being less if TCN was included in the medium. The mechanism of inhibition of DNA synthesis by TCN was shown not to involve DNA strand breakage or cross-linking, inhibition of polyamine biosynthesis, inhibition of purine de novo biosynthesis, inhibition of DNA polymerase alpha or DNA primase, or inhibition of ligation of Okazaki fragments. The effects of TCN on the incorporation of [3H]thymidine into Okazaki fragments and higher molecular weight DNA suggested the possibilities of inhibition of Okazaki fragment initiation and/or DNA polymerase delta.

Synthesis and preliminary evaluation of carbon-11-meta-hydroxyephedrine: a false transmitter agent for heart neuronal imaging.

Carbon-11-meta-hydroxyephedrine is a new radiotracer developed for mapping the sympathetic nerves of the heart. Carbon-11-meta-hydroxyephedrine is synthesized by direct N-methylation of metaraminol with [11C]methyl iodide in dimethyl formamide/dimethyl sulfoxide and purified by semi-preparative reversed-phase HPLC. Total synthesis time is 45 min from end-of-bombardment. Carbon-11-meta-hydroxyephedrine is produced in 40%-50% corrected radiochemical yield with a specific activity of 900 Ci/mmol. Routine radiochemical and chemical purity are 95% and 98%, respectively. Biodistribution studies in rats show high myocardial uptake. Pretreatment with desipramine, a drug known to selectively block neuronal uptake, results in a 92% decrease in tracer accumulation in the myocardium. Metabolic studies in guinea pigs show less than 5% metabolites in heart tissue 30 min after intravenous injection suggesting that [11C]meta-hydroxyephedrine is suitable for kinetic modeling. These preliminary results support this new tracer as a clinical agent for neuronal imaging of the heart.

Neuronal mapping of the heart with 6-[18F]fluorometaraminol.

The false neurotransmitter metaraminol labeled with fluorine-18 has been used to noninvasively assess regional adrenergic nerve density in the canine heart. Intravenous administration of 6-[18F]fluorometaraminol (FMR) results in high, selective accumulation of radioactivity in the heart; drug blocking studies with desipramine and reserpine confirm the neuronal locus of FMR. Iodine-125 labeled metaraminol, however, shows no selective accumulation in the canine heart. Positron emission tomography (PET) analyses with FMR of closed-chest dogs bearing left ventricular neuronal defects clearly delineate the region of neuronal impairment; blood perfusion in the left ventricle wall was homogeneous as determined by [13N]NH3 tomograms. The accumulation of FMR in regionally denervated dog heart correlates closely (r = 0.88) with endogenous norepinephrine concentrations. PET-generated 18F time-activity curves demonstrate marked kinetic differences between normal and denervated myocardium. FMR/PET analysis could be used to assess the heterogeneity of sympathetic innervation in human heart disease contingent on the development of FMR with sufficiently high specific activity to clearly avoid pressor activity.

Mouse brain distribution of a carbon-11 labeled vesamicol derivative: presynaptic marker of cholinergic neurons.

The regional mouse brain distribution of a new carbon-11 labeled derivative of vesamicol, [11C]-5-(N-methylamino)benzovesamicol [( 11C]MABV) is reported. Radiotracer concentrations in vivo are in the rank order of striatum greater than cortex greater than hippocampus greater than hypothalamus greater than cerebellum, consistent with reported distributions of other presynaptic cholinergic neuronal markers. In time course studies, striatum/cerebellum and cortex/cerebellum ratios for (-)-[11C]MABV continue to increase to values of 13 and 5, respectively, 75 min after i.v. injection of [11C]MABV. The specific binding in striatum and cortex is lowered by pretreatment with (+/-)-vesamicol, and shows stereoselectivity with lower uptake and lower ratios for the (+)-enantiomer. (-)-enantiomer. (-)-[11C]MABV is proposed as a positron-emitting radioligand for the in vivo study of presynaptic cholinergic neurons.

Synthesis of a high specific activity 125I-labeled analog of PK 11195, potential agent for SPECT imaging of the peripheral benzodiazepine binding site.

The peripheral benzodiazepine binding site ligand PK 11195 has been 125I-labeled by direct displacement of aromatic chlorine under solid-state conditions in 50-76% radiochemical yield and greater than 94% radiochemical purity. Purification by high pressure liquid chromatography increased the specific activity of the product from an initial 15-17 Ci/mmol to a final activity of 260-910 Ci/mmol. To determine the affinity of this [125I]PK 11195 analog for human glioma cells, saturation experiments were performed on monolayers of U251 human glioblastoma cells. Scatchard analysis of saturation data demonstrated that the [125I]PK 11195 analog binds to a single class of sites with a KD of 8.0 +/- 1.7 nM and maximal binding of 3.8 +/- 0.1 pmol/mg protein. These values are similar to those obtained when [3H]PK 11195 was assayed in U251 cells (KD = 14 +/- 3.4, Bmax = 4.1 +/- 1.3) suggesting that iodination does not appreciably alter the binding of PK 11195 to human glioma cells. In vivo autoradiographic studies of brain in C6 glioma bearing rats demonstrate selective binding of the radioligand to the tumor. These results suggest that this [125I]PK 11195 analog may be a useful radiotracer for the study of peripheral benzodiazepine binding sites.

Metabolic fate of the heart agent [18F]6-fluorometaraminol.

Studies were performed to determine whether [18F]6-fluorometaraminol (18F-FMR), a new neuronal heart radiopharmaceutical, is metabolized in vivo and if the metabolites are taken up in heart. Rat, dog, baboon and guinea pig were injected with 18F-FMR and tissue samples were analyzed for metabolites by HPLC. Liver contained the most metabolites of the tissues studied with 25-90% of the radioactivity present as metabolites at 1 h in all the species studied. While metabolites of 18F-FMR are found in blood, no significant accumulation of these metabolites is found in heart (less than or equal to 0.3%) 1 h after i.v. administration in any species except rat. These studies suggest that 18F-FMR is a suitable agent for quantitative imaging of the heart by positron emission tomography.

Radioiodinated benzodiazepines: agents for mapping glial tumors.

Two isomeric iodinated analogues of the peripheral benzodiazepine binding site (PBS) ligand Ro5-4864 have been synthesized and labeled in high specific activity with iodine-125. Competitive binding assays conducted with the unlabeled analogues indicate high affinity for PBS. Tissue biodistribution studies in rats with these 125I-labeled ligands indicate high uptake of radioactivity in the adrenals, heart, and kidney--tissues known to have high concentrations of PBS. Preadministration of the potent PBS antagonist PK 11195 blocked in vivo uptake in adrenal tissue by over 75%, but to a lesser degree in other normal tissues. In vivo binding autoradiography in brain conducted in C6 glioma bearing rats showed dense, PBS-mediated accumulation of radioactivity in the tumor. Ligand 6 labeled with 123I may have potential for scintigraphic localization of intracranial glioma.