[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.