Comparison of the pharmacological characteristics of rodent and marmoset GAT-1

F. Andreetta, S. Faedo, M. Negri, L. Mangiarini, M. Corsi & P. Wren, GlaxoSmithKline S.p.A., Psychiatry Centre of Excellence for Drug Discovery, Verona, Italy.

The selective targeting and modulation of the GABA transporter GAT-1, is of therapeutic interest in GABA-related CNS disorders, including epilepsy and anxiety. In the development of novel GAT-1 inhibitors it is important to understand possible species differences in the binding characteristics. [3H]Tiagabine binding experiments were therefore performed to obtain the pharmacological profiles of a range of known GAT-1 ligands in cortical brain membranes derived from animal species used in pre-clinical studies including rodents and non-human primates.

Membrane homogenates derived from the cerebral cortex of Sprague Dawley rats (Rattus norvegicus, 250-300g, male) and common marmosets (Callithrix jacchus, 300-400g, male) were prepared in ice-cold assay buffer (50mM Tris containing 1M NaCl and 5mM KCl, pH 7.7). Membrane protein (~10μg) was incubated for 2 hours at room temperature either with increasing concentrations of [3H]tiagabine (0.05nM-200nM) for saturation experiments or with 1nM [3H]tiagabine in the presence of varying concentrations of test compound for competition experiments. 100μM SKF-89976 (Soudijn et al., 2000) was used to determine non-specific binding. The association and dissociation (in the presence of 10μM cold tiagabine) of 5nM [3H]tiagabine , was determined using classical methodologies. The kinetic profile of tiagabine was also obtained by an adaptation of a method by Motulsky & Mahan (1984), in which the association of 5nM [3H]Tiagabine was measured in the presence of 30 nM of cold tiagabine. Bound and free radioactivity was obtained by filtration harvesting and quantified by liquid scintillation counting for analysis in GraphPad Prism®.

Saturation binding experiments revealed that tiagabine bound to a single site with a Kd of 29.4±2 nM on rat and 40±6 nM on marmoset membranes (n=3). Competition experiments revealed similar inhibitory constants for a range of GAT-1 ligands across the two species (Table 1, n=3; Soudijn et al., 2000). Additionally the kinetic parameters of tiagabine revealed similar kinetic profiles in both species. Classical kinetic experiments for tiagabine gave Kon and K off values of 1.89±0.23E+6 min-1M-1 and 0.043±0.003 min-1 respectively in rat and 2.24±0.38E+6 min-1 M-1 and 0.037±0.005min-1 respectively in marmoset (n=3). These data are in good agreement with data derived from the method of Motulsky & Mahan (1984), where tiagabine gave Kon and Koff values of 5.15±0.78E+6 min-1 M-1 and 0.038± 0.003 min-1 respectively in rat and 1.85±0.14E+6 min-1M -1 and 0.025 ±0.001 min-1 respectively in marmoset (n=3).

These studies are the first to report the characterisation of [3H]tiagabine binding in a non-human primate such as the common marmoset and agree well with those previously determined in the rat (Braestrup et al., 1990). We also observed a lack of any pharmacological differences between these two species.

Braestrup et al.,1990. J. Neurochem., 54(2):639-647.
Motulsky & Mahan,1984. Molecular Pharmacol., 25(1):1-9.
Soudijn et al.,2000. Current Med.Chem. 7,1063-1079.