G.K. Mulholland

In vivo Muscarinic Cholingeric Receptor Imaging in Human Brain with [11C]Scopolamine and Positron Emission Tomography

Cerebral muscarinic cholinergic receptors were imaged and regionally quantified in vivo in humans with the use of [11C]scopolamine and positron emission tomography. Previous studies in experimental animals have suggested the utility of radiolabeled scopolamine for in vivo measurements, on the bases of its maintained pharmacologic specificity following systemic administration and the exclusion of labeled metabolites from the brain. The present studies describe the cerebral distribution kinetics of [11C]scopolamine in normal subjects following intravenous injection. Scopolamine is initially delivered to brain in a perfusion-directed pattern. After 30 to 60 min, activity is lost preferentially from cerebral structures with low muscarinic receptor density including the cerebellum and thalamus. Activity continues to accumulate throughout a 2 h postinjection period in receptor-rich areas including cerebral cortex and the basal ganglia. The late regional concentration of [11C]scopolamine does not, however, accurately parallel known differences in muscarinic receptor numbers in these receptor-rich areas. Tracer kinetic analysis of the data, performed on the basis of a three-compartment model, provides receptor binding estimates in good agreement with prior in vitro measurements. Kinetic analysis confirms significant contributions of ligand delivery and extraction to the late distribution of [11C]scopolamine, reconciling the discrepancy between receptor levels and tracer concentration. Finally, a novel dual-isotope method for rapid chromatographic processing of arterial blood samples in radiotracer studies is presented. The combination of rapid chromatography and compartmental analysis of tracer distribution should have broad utility in future in vivo studies with short-lived radioligands.

Kinetic Evaluation of Positron-Emitting Muscarinic Receptor Ligands Employing Direct Intracarotid Injection

The development and characterization of new receptor ligands for in vivo binding assays are often both lengthy and expensive. It is therefore desirable to predict the suitability of a ligand early in the process of its evaluation. In the present study, compartmental analysis following intracarotid ligand injection in the monkey is used to evaluate the in vivo kinetics of the muscarinic cholinergic receptor antagonists [11C]tropanyl benzilate ([11C]TRB) and [11C]N-methylpiperidyl benzilate ([11C]NMPB). Animals were implanted with chronic subcutaneous access ports and indwelling catheters with tips located in the common carotid artery, just proximal to its bifurcation. The external carotid artery was ligated to ensure selective tracer delivery through the internal carotid artery to the brain. Positron emission tomography was used to measure brain tissue time–activity curves following tracer injections. CBF was estimated from the clearance of [15O]H2O, and receptor ligand distributions were analyzed according to a physiologic model consisting of an intravascular compartment and nonspecific plus free and receptor-bound tissue ligand compartments. In [11C]TRB studies, marked reductions in the forward ligand-receptor binding rate and in both the total and the specific binding tissue-to-plasma volumes of ligand distribution were observed after scopolamine receptor blockade or with low administered specific activity. Conversely, neither the distribution volume of the nonspecific plus free ligand compartment nor the rate of ligand dissociation from receptor sites was affected. In [11C]NMPB studies, tissue compartments describing specific binding and nonsaturable components could not be reliably separated. The receptor-related term in this case, the total tissue-to-plasma distribution volume, demonstrated reduction after low specific activity ligand injection. Comparison of the two ligands suggests that NMPB interacts more rapidly with the receptors and has a lower apparent volume of distribution than does TRB. Thus, NMPB may be the more suitable ligand if accurate estimates of binding dissociation rate are limited by temporal constraints or if simplified, one-tissue-compartment analyses are used. The carotid injection method appears promising for the initial evaluation of ligand kinetics, permitting physiologic compartmental analyses without measurement of input functions or chromatography of blood samples.

In Vitro and Ex Vivo Evaluation of Cyclic Aminoalkyl Benzilates as Potential Emission Tomography Ligands for the Muscarinic Receptor

A series of muscarinic antagonists were screened as potential receptor imaging agents. (+)2 alpha-tropanyl benzilate (TRB), N-methyl-4-piperidyl benzilate (NMPB) and several analogs amenable to labeling with positron emitting isotopes were evaluated for muscarinic binding to mouse brain tissue in vitro and ex vivo using [3H]quinuclidinyl benzilate as the probe. The in vitro assay directly compared the innate binding affinities of the compounds. The rank order of binding (IC50) was TRB (0.7 nm), QNB (0.8 nm), scopolamine (1.3 nm) and NMPB (1.6 nm). The ex vivo assay was used to gain information regarding the pharmacokinetics and brain penetration of the compounds in live animals. Ex vivo results demonstrated that TRB was rapidly taken up into the brain and was equipotent with QNB in occupying muscarinic binding sites at early time points, but TRB binding decreased twice as fast over time as QNB binding. The results suggest TRB would be a good candidate for radiolabeling and further study.

Evaluation of Quaternized and Neutral Muscarinic Receptor Ligands in Normal and DES-treated Rat

The localization of quaternized muscarinic receptor (mAChR) antagonists, [11C]methyl tropanyl benzilate ([11C]MTRB) and [11C]methyl quinuclidinyl benzilate ([11C]MQNB), in rat pituitary was compared to that of [11C]tropanyl benzilate ([11C]TRB), a neutral antagonist. The quaternized ligands localize via a mAChR-mediated mechanism as shown by 60% reduction in radioactivity concentrations in the presence of QNB. [11C]TRB appears to localize primarily by a non-mAChR specific mechanism. Induction of pituitary prolactinomas by diethylstilbestrol resulted in a reduction of [11C]MTRB pituitary localization compared to normals. Elevated serum prolactin levels due to prolactinoma presence had no measurable effect on myocardial [11C]MTRB uptake or on KD values. Bmax values for myocardial mAChR were similar for controls and for DES exposure of 10 weeks.