Michael A. Wallace

A novel radioligand for glycine transporter 1: characterization and use in autoradiographic and in vivo brain occupancy studies

INTRODUCTION In an effort to develop agents to test the NMDA hypofunction hypothesis of schizophrenia, benchmark compounds from a program to discover potent, selective, competitive glycine transporter 1 (GlyT1) inhibitors were radiolabeled in order to further study the detailed pharmacology of these inhibitors and the distribution of GlyT1 in brain. We here report the in vitro characterization of [35S](S)-2-amino-4-chloro-N-(1-(4-phenyl-1-(propylsulfonyl)piperidin-4-yl)ethyl)benzamide ([35S]ACPPB), a radiotracer developed from a potent and selective non-sarcosine-derived GlyT1 inhibitor, its use in autoradiographic studies to localize (S)-2-amino-6-chloro-N-(1-(4-phenyl-1-(propylsulfonyl)piperidin-4-yl)ethyl)benzamide (ACPPB) binding sites in rat and rhesus brain and for in vivo occupancy assays of competitive GlyT1 inhibitors. METHODS Functional potencies of unlabeled compounds were characterized by [14C]glycine uptake into JAR (human placental choriocarcinoma) cells and synaptosomes. Radioligand binding studies were performed with tissue homogenates. Autoradiographic studies were performed on tissue slices. RESULTS ACPPB is a potent (Kd=1.9 nM), selective, GlyT1 inhibitor that, when radiolabeled with [35S], is a well-behaved radioligand with low nondisplaceable binding. Autoradiographic studies of rat and rhesus brain slices with this ligand showed that specific binding sites were plentiful and nonhomogeneously distributed, with high levels of binding in the brainstem, cerebellar white matter, thalamus, cortical white matter and spinal cord gray matter. In vivo studies demonstrate displaceable binding of [35S]ACPPB in rat brain tissues following iv administration of this radioligand. CONCLUSIONS This is the first report of detailed anatomical localization of GlyT1 using direct radioligand binding, and the first demonstration that an in vivo occupancy assay is feasible, suggesting that it may also be feasible to develop positron emission tomography tracers for GlyT1.

Efficient utilization of [14C]carbon dioxide as a phosgene equivalent for labeled synthesis

Abstract Addition of one equivalent [14C]carbon dioxide to primary or secondary amines in the presence of ternary base, followed by reaction of the resulting [14C]carbamate salt with phosphorus oxychloride or thionyl chloride, represents a cost effective alternative to labeled phosgene.

Identification and characterization of a selective radioligand for melanin-concentrating hormone 1-receptor (MCH1R).

We have developed and characterized [(35)S]4a as a potent and selective radioligand for melanin-concentrating hormone 1-receptor (MCH1R). Compound [(35)S]4a showed appreciable specific signals in brain slices prepared from wild-type mice but not from MCH1R deficient mice, confirming the specificity and utility of [(35)S]4a as a selective MCH1R radioligand for ex vivo receptor occupancy assays.

Absorption, metabolism, and excretion of [14C]mk-0767 (2-methoxy-5-(2,4-dioxo-5-thiazolidinyl)-N[[4-(trifluoromethyl)phenyl] methyl]benzamide) in humans

MK-0767 (KRP-297; 2-methoxy-5-(2,4-dioxo-5-thiazolidinyl)-N-[[4-(trifluoromethyl)phenyl] methyl]benzamide) is a thiazolidinedione (TZD)-containing dual agonist of the peroxisome proliferator-activated receptors α and γ that has been studied as a potential treatment for patients with type 2 diabetes. The metabolism and excretion of [14C]MK-0767 were evaluated in six human volunteers after a 5-mg (200 μCi) oral dose. Excretion of 14C radioactivity was found to be nearly equal into the urine (∼50%) and feces (∼40%). Elimination of [14C]MK-0767 was primarily by metabolism, with minimal excretion of parent compound into the urine (<0.5% of dose) and feces (∼14% of the dose). [14C]MK-0767 was the major circulating compound-related entity (>96% of radioactivity) through 48 h postdose. It was also found that ∼91% of the total radioactivity area under the curve was due to intact MK-0767. Several minor metabolites were detected in plasma (<1% of radioactivity, each), formed by cleavage of the TZD ring and subsequent S-methylation and oxidation. All the metabolites excreted into urine were formed by TZD cleavage, whereas the major metabolite in feces was the O-demethylated derivative of MK-0767.

The synthesis of HIV reverse transcriptase inhibitors [14C]L-697,661, [14C]L-697,639, [14C]L-702,007

The synthesis of three carbon-14 labeled reverse transcriptase inhibitors has been accomplished by elaboration of a common intermediate, 5-ethyl-6-methyl-3-amino-2-(1H)-[4- 14 C] -pyridinone (7). Ethyl [l- 14 C]formate, prepared by esterification of sodium [ 14 C]formate, was combined with 2-pentanone under basic conditions to afford 3-[ 14 C]carboxaldehyde-2-pentanone sodium salt (2). The pyridinone ring was constructed by condensation of 2 with nitro acetamide 4. Reduction of the nitro group afforded 5-ethyl-6-methyl-3-amino-2-(1H)-[4- 14 C]-pyridinone (7)(specific activity 54 mCi/mmol). Subsequent alkylation of 7 provided the desired reverse transcriptase inhibitors [ 14 C]L-697,661, [ 14 C]L-697,639. and [ 14 C]L-702.007.

Synthesis, stability, and radiolytic decomposition of carbon‐14 labeled MK0677

MK0677 is an orally active growth hormone secretagogue. The crystallized carbon-14 labeled material was found to undergo radiolytic decomposition via a peroxide intermediate which resulted in loss of the benzyl group. The rate was diminished when the tracer was crystallized from nitrogen-degassed solvents. Storage stability was best in aqueous ethanol.