Molecular targets versus models for new antiepileptic drug discovery

Abstract

Animal models have played a key role in the discovery and characterization of all marketed antiepileptic drugs (AED). The conventional wisdom is that the standard animal screening models are becoming obsolete because they fail to identify compounds that act in mechanistically new ways and as a result do not offer therapeutic advantages over presently available agents. In fact, far from only detecting me-too drugs, the models often uncover compounds with distinctive profiles of activity in various types of epilepsy and in addition have unexpected efficacy in non-epilepsy conditions, such as neuropathic pain, bipolar disorder, and migraine. Moreover, the animal models-because they are unbiased with respect to mechanism-provide an opportunity to uncover drugs that act in new ways and through new targets, such as α2δ and SV2A. In vitro testing is not likely to replace screening in animal models because in vitro systems cannot model the specific pharmacodynamic actions required for seizure protection, and do not assess bioavailability and brain accessibility.

Inactivation of the Constitutively Active Ghrelin Receptor Attenuates Limbic Seizure Activity in Rodents

“…The ghrelin mimetic capromorelin and the presumed GHSR antagonist A778193 (found in this study to be a GHSR inverse agonist) were generously provided by Dr. Luc Ver Donck and Dr. Dieder Moechars. The ghrelin fragments (1-5) amide, (1-7), (1-9), (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11), (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14), and (23-28) were supplied by Phoenix Pharmaceuticals (Karlsruhe, Germany). All other chemicals were analytical reagent grade or better and were supplied by Merck (Darmstadt, Germany).…”

“…Cells were subcultured at 70 to 80 % confluency and re-suspended in 96-well plates at 60,000 cells per well. Concentrations of A778193 were added in quadruplicate to the cells and incubated for 90 minutes at 37°C in air/CO 2 (1-7), (1-9), (1-11), (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14), and (23)(24)(25)(26)(27)(28) were tested next to the GHSR agonist capromorelin. A sigmoidal dose response curve was fitted using GraphPad Prism software, GraphPad Software, (La Jolla, USA), yielding a pEC 50 and curve maximum.…”

“…As previously described for activation, cells were first loaded with Fluo-4-acetoxymethyl ester and then pre-incubated for 3 minutes with ghrelin, capromorelin, ghrelin (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) or ghrelin (1-5) amide to induce desensitization. Three minutes later, a second stimulation with ghrelin was performed, and it consisted of a concentration response curve for ghrelin (range, 10 -6 -10 -10 M).…”

“…After pretreatment with ghrelin at 10 -6 M, 10 -7 M, or 10 -8 M for the duration of 3 minutes, the cells were washed and re-stimulated with ghrelin (dose response 10 -6 M -10 -10 M) after 3 minutes, 20 minutes, 1 h, 2 h , 5 h, or 24 h, respectively. e In vitro desensitization concentration response curve (3 minutes) for ghrelin, the GHSR agonist capromorelin, ghrelin (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14), and ghrelin(1-5) amide. Ghrelin (1)(2)(3)(4)(5) amide has a much lower capacity to desensitize the GHSR compared to ghrelin and capromorelin.…”

“…Thus, the search for new antiepileptic drugs (AEDs) is ongoing, and the emphasis is being placed in the discovery of compounds that have distinct profiles of activity than the AEDs currently on the market [13]. Increasing attention is given toward neuropeptides as potential leads in preventing seizures since they are less likely to fall under the category of "me-too" AEDs [14].…”