TPA023 [7-(1,1-Dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine], an Agonist Selective for α2- and α3-Containing GABA_AReceptors, Is a Nonsedating Anxiolytic in Rodents and Primates

Abstract

7-(1,1-Dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-1,2,4-triazolo [4,3-b]pyridazine (TPA023) is a triazolopyridazine that binds with equivalent high (subnanomolar) affinity to the benzodiazepine binding site of recombinant human GABA A receptors containing an ␣1, ␣2, ␣3, or ␣5 subunit but has partial agonist efficacy at the ␣2 and ␣3 subtypes and essentially antagonist efficacy at the ␣1 and ␣5 subtypes. In rats, TPA023 gave time-and dose-dependent occupancy after oral dosing, with 50% occupancy corresponding to a dose of 0.42 mg/kg. It has anxiolytic-like activity in unconditioned (elevated plus maze) and conditioned (fear-potentiated startle and conditioned suppression of drinking) rat models of anxiety with minimum effective doses (MED; 1-3 mg/kg) corresponding to 70 to 88% occupancy. However, there was no appreciable sedation in a response sensitivity (chain-pulling) assay at a dose of 30 mg/kg, resulting in 99% occupancy. Similarly, TPA023 was robustly anxiolytic in the squirrel monkey conditioned emotional response assay, with a MED of 0.3 mg/kg, but did not produce any sedation in a lever-pressing test of sedation even at 10 mg/kg. TPA023 produced no impairment in performance in the mouse Rotarod assay, and there was only a mild interaction with ethanol. In addition to anxiolytic-like efficacy, TPA023 had anticonvulsant activity in a mouse pentylenetetrazole seizure model. Finally, TPA023 did not cause precipitated withdrawal in mice treated for 7 days with the nonselective agonist triazolam, nor did N-methyl-␤-carboline-3-carboxamide (FG 7142) precipitate withdrawal in mice treated for 7 days with TPA023. In summary, the novel ␣2/␣3-selective efficacy profile of TPA023 translates into a nonsedating anxiolytic profile that is distinct from nonselective agonists.GABA A receptors are ligand-gated chloride ionophores that mediate the fast synaptic as well as tonic extrasynaptic inhibitory effects of GABA (Mody and Pearce, 2004). They are pentameric assemblies of proteins (Cromer et al., 2002) derived from a family of 16 genes (␣1-6, ␤1-3, ␥1-3, ␦, ⑀, , and ; Simon et al., 2004). Despite the theoretically large number of possible pentameric permutations that can arise from these 16 genes, only relatively few combinations are found within the central nervous system (McKernan and Whiting, 1996). Most of these native receptors contain ␣, ␤, and ␥ subunits in a 2:2:1 stoichiometry with an alternating ␣␤␣␤␥ arrangement as viewed from the synapse (Minier and Sigel, 2004).In addition to binding sites for their endogenous ligand, GABA A receptors also possess recognition sites for a number of pharmacologically active classes of compounds, including

Are GABAA Receptors Containing α5 Subunits Contributing to the Sedative Properties of Benzodiazepine Site Agonists?

“…Specifically, sedative effects of BZs were principally attributed to a 1 -containing GABA A receptor subtypes, anxiolytic actions to the a 2 -/a 3 -containing receptors, anterograde amnesic effects to the a 1 /a 5 subtypes, anticonvulsant activity partially to the a 1 -containing receptors, and muscle relaxant effects largely to a 2 -containing receptors (Atack et al, 2006a;Low et al, 2000;McKernan et al, 2000;Rudolph and Möhler, 2006;Rudolph et al, 1999). Moreover, it appears that L-838417, a ligand with zero efficacy at the a 1 subtype, is unable to engender sedation in rodents or monkeys , and similar conclusions were drawn from behavioral experiments with other compounds functionally selective for receptors other than a 1 -containing receptors (Atack et al, 2006c;Dias et al, 2005;Licata et al, 2005). Since the a 1 -subunit is critically involved in much of the unwanted psychomotor activity of nonselective BZs, 'a 1 -neutral' agonists at BZ site are being pursued in the quest for anxioselective anxiolytics (Basile et al, 2004;Whiting, 2006).…”

“…A close congener of that compound was anxiolytic in the rat EPM test at 3 mg/kg, whereas it showed no significant activity in a surrogate assay of sedation (chain-pulling test in rats) and in the mouse rotarod test when tested in doses up to 10 mg/kg; reportedly, this compound was chosen as a clinical candidate . Similarly, a 1,2,4-triazolo [4,3-b]pyridazine, TPA023, exerted anxiolytic-like activity in the rat EPM and fearpotentiated startle tests at the dose of 1 mg/kg as well as in the assay of conditioned suppression of drinking at 3 mg/kg, whereas, at doses up to 30 mg/kg, it did not significantly affect behavior of rats in the chain-pulling test nor rotarod performance of mice (Atack et al, 2006c). However, the same experimental group, when testing pagoclone, detected sedative action of this BZ site agonist dosed at 1 mg/kg in the chain-pulling test, while already at 0.3 mg/kg in the SLA test (Atack et al, 2006b).…”

“…On the other hand, it is noticeable that standard studies of motor activity, which form the very basis of knowledge of drug effects on behavior (Kelley, 1993), were not employed for characterization of recently reported putative anxioselective compounds (Atack et al, 2006c;Jennings et al, 2006;Griebel et al, 2001;Russell et al, 2006). Jennings et al (2006) reported on an a2/a3-BZ site-selective ligand from the class of imidazo[1,2-b][1,2,4]triazines with anxiolytic activity in the rat EPM test at the dose of 1 mg/kg, which showed no motor incapacitation in the mouse rotarod test at doses up to 30 mg/kg.…”

“…To date, all apparently non-sedating BZ site ligands, which are devoid of activity at a 1 -containing GABA A receptors, engendered essentially antagonist (Atack et al, 2006c;Dias et al, 2005) or only partial agonist efficacy at a 5 -containing GABA A receptors (Griebel et al, 2001;McKernan et al, 2000). Therefore, it cannot be ruled out that substantial efficacy at a 5 -containing GABA A receptors may contribute to sedative effects.…”

Combining Sanford Arylations on Benzodiazepines with the Nuisance Effect

“…The current and previous library of benzodiazepines (Scheme 1) was tested for GABA binding 8. None of the current benzodiazepines displayed any appreciable biological activity although 7‐chloro‐benzodiazepines, as expected, had reasonable activity, although were ca .…”