Mark E. Powell

[11C]AZ10419369: a selective 5-HT1B receptor radioligand suitable for positron emission tomography (PET). Characterization in the primate brain.

The 5-HT1B receptor has been implicated in several psychiatric disorders and is a potential pharmacological target in the treatment of depression. Here we report the synthesis of a novel PET radioligand, [11C]AZ10419369 (5-methyl-8-(4-methyl-piperazin-1-yl)-4-oxo-4H-chromene-2-carboxylic acid (4-morpholin-4-yl-phenyl)-amide), for in vivo visualization of 5-HT1B receptors in the brains of macaques and humans subjects. [11C]AZ10419369 was prepared by N-methylation of (8-(1-piperazinyl)-5-methylchrom-2-en-4-one-2-(4-morpholinophenyl) carboxamide, using carbon-11 methyl triflate. Regional brain uptake patterns of [11C]AZ10419369 were characterized by PET measurements in two macaques and a preliminary study in two human subjects. In addition, AZ10419369 was prepared in tritium labeled form for in vitro autoradiography studies in macaque brain tissue sections. The radiochemical purity of [11C]AZ10419369 was >99% and specific radioactivity was >3600 Ci/mmol. After iv injection of [11C]AZ10419369, 3-4% was in brain after 7.5 min. The regional brain distribution of radioactivity was similar in humans and macaques showing the highest uptake of radioactivity in the occipital cortex and the basal ganglia, in accord with autoradiographic studies performed using [3H]AZ10419369. Uptake was moderate in the temporal and frontal cortical regions, lower in the thalamus and lowest in the cerebellum. In macaques pre-treated with the selective 5-HT1B receptor antagonist, AR-A000002, binding was reduced in a dose-dependent manner, consistent with specific binding to 5-HT1B receptors. These data support [11C]AZ10419369 as a suitable radioligand for labeling 5-HT1B receptors in the primate brain. This radioligand may be useful in future studies evaluating drug-induced receptor occupancy and measurement of brain 5-HT1B receptor levels in patients with psychiatric disorders.

[N-methyl-3H3]AZ10419369 Binding to the 5-HT1B Receptor: In Vitro Characterization and in Vivo Receptor Occupancy

Radiotracers suitable for positron emission tomography studies often serve as preclinical tools for in vivo receptor occupancy. The serotonin 1B receptor (5-HT1B) subtype is a pharmacological target used to discover treatments for various psychiatric and neurological disorders. In psychiatry, 5-HT1B antagonists may provide novel therapeutics for depression and anxiety. We report on the in vitro and in vivo evaluation of tritiated 5-methyl-8-(4-methyl-piperazin-1-yl)-4-oxo-4H-chromene-2-carboxylicacid (4-morpholin-4-yl-phenyl)-amide ([N-methyl-3H3]AZ10419369), a potent 5-HT1B radiotracer. [N-methyl-3H3]-AZ10419369 showed saturable single-site high-affinity in vitro binding (guinea pig, Kd = 0.38 and human, Kd = 0.37) to guinea pig or human 5-HT1B receptors in recombinant membranes and high-affinity (Kd = 1.9 nM) saturable (Bmax = 0.099 pmol/mg protein) binding in membranes from guinea pig striatum. When [N-methyl-3H3]AZ10419369 was administered to guinea pigs by intravenous bolus, the measured radioactivity was up to 5-fold higher in brain areas containing the 5-HT1B receptor (striatum/globus pallidus, midbrain, hypothalamus, and frontal cortex) compared with the cerebellum, the nonspecific binding region. Specific uptake peaked 30 min after injection with slow dissociation from target regions, as suggested by the in vitro binding kinetic profile. Pretreatment with 6-fluoro-8-(4-methyl-piperazin-1-yl)-4-oxo-4H-chromene-2-carboxylic acid [4-(4-propionyl-piperazin-1-yl)-phenyl]-amide (AZD1134) and 2-aminotetralin (AR-A000002), 5-HT1B-selective ligands, inhibited [N-methyl-3H3]AZ10419369-specific binding in a dose-dependent manner. In the guinea pig striatum, AZD1134 (ED50 = 0.017 mg/kg) occupies a greater percentage of the 5-HT1B receptors at a lower administered dose than AR-A000002 (ED50 = 2.5 mg/kg). In vivo receptor occupancy is an essential component to build binding-efficacy-exposure relationships and compare novel compound pharmacology. [N-methyl-3H3]AZ10419369 is a useful preclinical tool for investigating 5-HT1B receptor occupancy for novel compounds targeting this receptor.

Contribution of Artifacts to N-Methylated Piperazine Cyanide-Adduct Formation in vitro From N-Alkyl Piperazine Analogs

In the liver microsome cyanide (CN)-trapping assays, piperazine-containing compounds formed significant N-methyl piperazine CN adducts. Two pathways for the N-methyl piperazine CN adduct formation were proposed: 1) The α-carbon in the N-methyl piperazine is oxidized to form a reactive iminium ion that can react with cyanide ion; 2) N-dealkylation occurs followed by condensation with formaldehyde and dehydration to produce N-methylenepiperazine iminium ion, which then reacts with cyanide ion to form the N-methyl CN adduct. The CN adduct from the second pathway was believed to be an artifact or metabonate. In the present study, a group of 4′-N-alkyl piperazines and 4′-N-[13C]methyl–labeled piperazines were used to determine which pathway was predominant. Following microsomal incubations in the presence of cyanide ions, a significant percentage of 4′-N-[13C]methyl group in the CN adduct was replaced by an unlabeled natural methyl group, suggesting that the second pathway was predominant. For 4′-N-alkyl piperazine, the level of 4′-N-methyl piperazine CN adduct formation was limited by the extent of prior 4′-N-dealkylation. In a separate study, when 4′-NH-piperaziens were incubated with potassium cyanide and [13C]-labeled formaldehyde, 4′-N-[13C]methyl piperazine CN-adduct was formed without NADPH or liver microsome suggesting a direct Mannich reaction is involved. However, when [13C]-labeled methanol or potassium carbonate was used as the one-carbon donor, 4′-N-[13C]methyl piperazine CN adduct was not detected without liver microsome or NADPH present. The biologic and toxicological implications of bioactivation via the second pathway necessitate further investigation because these one-carbon donors for the formation of reactive iminium ions could be endogenous and readily available in vivo.