G. Keith Mulholland

[11C]Tropanyl Benzilate—Binding to Muscarinic Cholinergic Receptors: Methodology and Kinetic Modeling Alternatives:

Quantitative estimation of cerebral muscarinic receptors was investigated with the use of the antagonist [11C]tropanyl benzilate ([11C]TRB) and positron emission tomography (PET). Kinetic modeling alternatives were examined with the goal of identifying an analysis method providing stable receptor measures, yet avoiding biases from inappropriate reductions in model complexity. Dynamic PET scans were performed on six young normal volunteers. Several modeling approaches yielding relative receptor density measures were evaluated: (a) a single “late” scan using relative tracer concentration values; (b) a slope estimate from graphic analysis (Patlak plot); (c) a two-compartment, two-parameter model (transport and total ligand distribution volume); (d) a three-compartment, two-parameter model using the free + nonspecific distribution volume, DV‘, fixed to the cerebellar value; (e) an early scan for transport, a fixed value for DV’, and a single late scan for the binding rate constant; and (f) a three-compartment, three-parameter model. Both computer simulations and PET scan results indicate all methods provide receptor density index measures with the same rank order as in vitro measures. Oversimplified approaches (methods 1 and 2) yield a more highly nonlinear relation between the estimated receptor density index and the known receptor density than do methods retaining greater model complexity (methods 3–6). However, noise propagation into the receptor measure is greater for the more complex methods. Reliable receptor density information can be obtained from kinetic [11C]TRB PET studies, with methods 3–5 providing the most appropriate levels of model complexity for estimates of relative muscarinic receptor density.

[18F]fluoroethoxy-benzovesamicol, a PET radiotracer for the vesicular acetylcholine transporter and cholinergic synapses

Loss of cholinergic transmission in the cortex and hippocampus is a characteristic feature of Alzheimers disease, and visualization of functional cholinergic synapses in the brain with PET could be a useful method for studying this degenerative condition in living humans. We investigated [18F]fluoroethoxybenzovesamicol, (−)‐[18F]FEOBV, (−)‐(2R,3R)‐trans‐2‐hydroxy‐3‐(4‐phenylpiperidino)‐5‐(2‐[18F]fluoroethoxy)‐1,2,3,4‐tetralin, a high affinity positron emitting ligand for the vesicular acetylcholine transporter, as a potential in vivo cholinergic synapse mapping agent. Rodent biodistribution, dosimetry, stereospecificity of biological effects, pharmacologic blocking studies, in vivo rodent brain autoradiography and metabolites were examined. (−)‐[18F]FEOBV brain uptake following intravenous injection was robust, with 2.65% dose/brain in mice at 5 min, and the regional localization matched the known distributions of presynaptic cholinergic markers at later times. Both the cholinergic localization and curare‐like effects of FEOBV were associated with the “(−)”‐enantiomer exclusively. (−)‐[18F]FEOBV regional brain distribution in rodents was changed little by pretreatment with haloperidol, (+)‐3‐PPP, or E‐2020, indicating FEOBV, unlike other vesamicol analogs, did not interact in vivo with dopamine or σ receptor systems. Autoradiography of rat brain 3 h following i.v. injection of (−)‐[18F]FEOBV showed high localization in brain areas rich in presynaptic cholinergic elements. Metabolic defluorination in rodents was modest, and analysis of brain tissue following tracer administration found FEOBV as the only extractable radioactive species. (−)‐[18F]FEOBV dosimetry calculated from rat data estimate 10 mCi doses can be given to humans. These studies show FEOBV maps cholinergic areas with high specificity in vivo, and may provide a noninvasive means to safely and accurately gauge the functional integrity of cholinergic synapses in man using PET. Synapse 30:263–274, 1998.

In Vivo Quantification of Cerebral Muscarinic Receptors in Normal Human Aging Using Positron Emission Tomography and [11C]Tropanyl Benzilate

Regional cerebral muscarinic cholinergic receptor binding was quantified in normal young and elderly subjects employing the muscarinic antagonist radioligand [11C]tropanyl benzilate (TRB). Binding was determined by kinetic analyses of positron emission tomographic (PET) determinations of cerebral activity in conjunction with radial arterial blood sampling following intravenous radiotracer injection. A significant, but minor (8%), loss of frontal cortical receptors relative to whole brain average receptor density was found with advancing age. Parametric estimates of binding suggest small reductions in cerebral cortex binding as well as increases in brain stem and cerebellar binding underlying the observed pattern difference. However, these latter changes did not achieve statistical significance. We conclude that cerebral muscarinic receptor availability, as depicted by antagonist binding, does not undergo a major decline during normal aging of the adult human brain. The cerebral cortical cholinergic dysfunction in elderly subjects, suggested by prior clinical evidence, is not attributable to major loss of total muscarinic cholinoceptive capacity.

Quantification of Muscarinic Cholinergic Receptors with [11C]NMPB and Positron Emission Tomography: Method Development and Differentiation of Tracer Delivery from Receptor Binding

Quantification of human brain muscarinic cholinergic receptors was investigated with the use of [11C]N-methyl-4-piperidyl benzylate (NMPB) and positron emission tomography (PET). Whole-brain uptake of NMPB at 90 to 110 minutes after intravenous injection was approximately 10% of the administered dose. The initial cerebral distribution of NMPB corresponded to the pattern of cerebral perfusion; however, at progressively longer postinjection intervals, regional distinctions consistent with muscarinic receptor binding were evident: activity at 90 to 110 minutes postinjection was highest in the striatum and cerebral cortex, intermediate in the thalamus and pons, and lowest in the cerebellum. After the development of a chromatographic system for isolation of authentic [11C]NMPB in plasma, tracer kinetic modeling was used to estimate receptor binding from the cerebral and arterial plasma tracer time-courses. Ligand transport rate and receptor-binding estimates were obtained with the use of compartmental models and analytical methods of varying complexity, including a two-parameter pixel-by-pixel-weighted integral approach and regional least-squares curve-fitting analyses employing both two-and three-compartment model configurations. In test—retest experiments, precision of the methods and their abilities to distinguish altered ligand delivery from binding in occipital cortex during an audiovisual presentation were evaluated. Visual stimulation increased the occipital blood-to-brain NMPB transport rate by 25% to 46% in estimates arising from the various approaches. Weighted integral analyses resulted in lowest apparent transport changes and in a concomitant trend toward apparent binding increases during visual activation. The regional least-squares procedures were superior to the pixel-by-pixel method in isolating the effects of altered tracer delivery from receptor-binding estimates, indicating larger transport effects and unaltered binding. Precision was best (less than 10% test—retest differences) for the weighted integral analyses and was somewhat lower in the least-squares analyses (10–25% differences). The authors conclude that pixel-by-pixel-weighted integral analyses of NMPB distribution introduce transport biases into receptor-binding estimates. Similar confounding effects also are predicted in noncompartmental analyses of delayed radiotracer distribution. The use of regional nonlinear least-squares fitting to two- and three-compartment models, although more labor intensive, provides accurate distinction of receptor-binding estimates from tracer delivery with acceptable precision in both intra- and intersubject comparisons.

Synthesis, in vivo biodistribution and dosimetry of [11C]N-Methylpiperidyl benzilate ([11C]NMPB), a muscarinic acetylcholine receptor antagonist

4-N-Methylpiperidyl benzilate (NMPB), a high affinity antagonist for the muscarinic cholinergic receptor, has been synthesized in carbon-11-labeled form through the N-[11C]methylation of 4-piperidylbenzilate. The product was isolated by HPLC, and obtained in yields (> 100 mCi) and specific activities (500-3000 Ci/mmol) sufficient for in vivo evaluation in small animals. Time-dependent regional brain distributions in rats and mice showed high radiotracer uptake and retention in striatum and cortex, and low in cerebellum, consistent with muscarinic cholinergic receptor distributions. Radiotracer retention in tissues could be significantly reduced by pretreatment of animals with a large dose of a competing antagonist, quiniclidinyl benzilate. Whole body biodistribution in rats was used to calculate the expected human internal radiation dosimetry for this new radiopharmaceutical. These animal experiments formed the basis for subsequent introduction of [11C]NMPB into human use with positron emission tomography.

Direct simultaneous production of [15O]water and [13N]ammonia or [18F]fluoride ion by 26 MeV proton irradiation of a double chamber water target

A simple double liquid chamber target was developed to provide the option for simultaneous production of [15O]H2O and either 13N or 18F using a single proton beam. Irradiation of natural water in a thin aluminium front chamber produced [15O]H2O by the 16O(p, pn)15O reaction directly. Large (0.5-1.0 Ci) doses of sterile [15O]H2O (greater than 99.95% radionuclide purity) were routinely prepared in 1 min from end of 20 microA bombardments using this target and an in-line mixed bed ion exchange column purification. Water in the thin front chamber degraded proton energies on exit to 20-18 MeV. The rear silver liquid chamber was threefold thick to 17 MeV protons in water and it efficiently produced either 13N by the 16O(p, alpha)13N reaction or [18F]fluoride ion by the 18O(p, n)18F reaction. Both target chambers were overpressurized with at least 6 atm of gas to minimize boiling/cavitation of water at high beam currents. Using hydrogen as the overpressure gas on the back chamber and an in-line anion exchange column radionuclidic cleanup process, high yields of sterile, aqueous [13N]NH3 (40-200 mCi; 20 microA) were produced directly from the back chamber at the same time that [15O]H2O was being produced from the front chamber. The combination of this target system with a cyclotron capable of generating 26-30 MeV protons provides great flexibility and simplicity for rapid, high volume production of the three best validated and most widely used radiopharmaceuticals at the present time in clinical positron emission tomography: [15O]H2O, [13N]NH3 and [18F]FDG.

Evaluation of ( − )[18F]fluoroethoxybenzovesamicol as a new PET tracer of cholinergic neurons of the heart

18F-labeled (-)fluoroethoxybenzovesamicol [(-)FEOBV] is a novel PET tracer which binds to the vesicular acetylcholine transporter of cholinergic neurons. To evaluate the in vivo binding specificity and kinetic properties of (-)FEOBV, studies were performed in isolated working rat hearts. External gamma,gamma-coincidence monitoring of hearts indicated high extraction of radiotracer by the myocardium and rapid wash-out of unbound tracer (> 90% of maximal accumulation) within 5 min. Inclusion of (-)vesamicol (10 microM) throughout perfusion decreased the retention of (-)FEOBV by 71% (P < 0.005) and 76% (P < 0.005) in atria and ventricles, respectively. However, the initial uptake rate of the tracer was unaffected. Additional experiments showed the inactive stereoisomer, (+)FEOBV, to have a lower retention than the (-)FEOBV isomer in ventricles, indicating stereospecificity of the binding process that is consistent with structure-activity relationships of vesamicol congeners. The results indicate (-)FEOBV to be a moderately specific probe of vesamicol-sensitive binding in cholinergic neurons of the heart in experimental conditions that assure adequate washout of unbound tracer. However, the utility of the radiotracer for in vivo studies with PET is likely to be limited by the low rate of specific binding in myocardium consistent with the low density of cholinergic neurons in the heart.

Routine synthesis of N-[11C-methyl]scopolamine by phosphite mediated reductive methylation with [11C]formaldehyde

A synthesis of [11C]scopolamine capable of clinical delivery of this agent in high specific activity is described. The precursor [11C]formaldehyde was produced by catalytic oxidation of [11C]CH3OH over metallic silver and was used to N-11C-methylate norscopolamine using aqueous neutral potassium phosphite as the reducing agent. The labeling reaction was complete after 5 min at 75-80 degrees C and the [11C]scopolamine (99% radiochemical purity) was isolated by preparative HPLC. Total synthesis time is less than 45 min. Decay corrected radiochemical yields from [11C]CO2 are presently 20-43%.

Multiphase extraction: rapid phase-transfer of [18F]fluoride ion for nucleophilic radiolabeling reactions.

In multiphase extraction [18F]fluoride ion for radiolabeling is recovered from target water by passage through a small column of microporous polymer impregnated with a lipophilic cryptand or quaternary ammonium salt. The 18O enriched water can be recovered for reuse. The [18F]fluoride ion-pair is eluted from the column by a small volume of acetonitrile or other organic solvent. Evaporation of the acetonitrile removes traces of water to yield a reactive ion pair for nucleophilic radiofluorination reactions. A wide range of ion-pairs based on K+ or NH+4 cryptands or quaternary ammonium salts can be employed. The method was applied to the synthesis of [18F]FDG.

Regional brain distribution of [18F]GBR 13119, a dopamine uptake inhibitor, in CD-1 and C57BL/6 mice.

We have examined the regional brain distribution of [18F]GBR 13119 (18F: beta +, T1/2 = 110 min), a dopamine uptake inhibitor, in CD-1 and C57BL/6 mice. High levels of binding are observed in the striatum of both species, with striatum/cerebellum ratios of 3-4 at 60 min after injection of the radiotracer. Striatum radioactivity and striatum/cerebellum ratios are more than 50% reduced in C57BL/6 mice treated chronically with the neurotoxin MPTP. We conclude mice are an appropriate model for the in vivo study of the dopamine uptake system, and that [18F]GBR 13119 may be a suitable in vivo marker for degeneration of striatal dopaminergic neurons.