Modulation of the Kappa Opioid System in TLE

With a prevalence of 1-2% worldwide, epilepsy is one of the most frequent neurological diseases affecting people of all ages. Of the 870 million people living in the European Region, over 5 million suffer from epilepsy. Current pharmacotherapies are not sufficiently effective in over 30 % of patients and cause severe side effects in 10% to 90% of people. Therefore, research efforts are needed towards overcoming the limitations of present therapies, with the final goal to improve treatment efficacy, patient compliance and to reduce complications. Recent evidence provided by us supports the idea of targeting the kappa opioid receptor (KOR) with molecules activating the G- protein signaling pathway preferentially over the ß-arrestin2 signaling to achieve anti-ictal effects with low adverse effects. The idea of so-called biased agonists has emerged over the recent years based on a deeper understanding of G-protein coupled receptors and is supported by computer-aided modeling. The overall goal of this research proposal is to advance the understanding of the contribution of the Dynorphin/KOR system in pathophysiological processes of TLE, that can be used as a molecularly defined target and therapeutic strategy to modulate seizures and progressive neuropathological changes associated with TLE. The present project comprises basic research as experimental work undertaken (i) to acquire new insights and better knowledge in the modulation of the dynorphin/KOR system in temporal lobe epilepsy (TLE), and the underlying mechanism responsible for separation of anti-ictal effects of KOR activation from adverse effects (aversion, motor impairment) -clarification of the pharmacological and neurobiochemical background of this separation is essential for an effective therapy of TLE with reduced side effects; (ii) to identify and characterize scientifically proven novel KOR ligands from the class of diphenethylamines with target-oriented pharmacology (i.e. G-protein biased KOR agonists), efficacy and favorable safety profile for the treatment of TLE patients, and (iii) to assess the functional selectivity, and structural and molecular determinants that can form the basis for an improvement of the benefit/risk index. For this project, three expert teams from the two universities in Innsbruck and the MaxPlanckInstitute of Biochemistry in Martinsried will combine their expertise.. The outcome of this work will expand the understanding on ligand-receptor interaction, molecular mode of action and KOR-mediated signaling pathways, and ultimately may be instrumental to the design and development of safer, better tolerated and more efficacious drugs for therapeutic use in humans.

With a prevalence of 1-2% worldwide, epilepsy is one of the most frequent neurological diseases affecting people of all ages. Of the 870 million people living in the European Region, over 5 million suffer from epilepsy. Current pharmacotherapies are not sufficiently effective in over 30 % of patients and cause severe side effects. Therefore, research efforts are needed towards overcoming the limitations of present therapies, with the final goal to improve treatment efficacy and patient compliance. Recent evidence supports the idea of targeting the kappa opioid receptor (KOPr) with molecules activating the G-protein signaling pathway preferentially over the -arrestin2 signaling to achieve anti-ictal effects with low adverse effects. The idea of so-called "biased agonists" has emerged over the recent years based on a deeper understanding of G-protein coupled receptors. Within this project we characterized a number of KOPr ligands from the class of diphenethylamines in-vitro and in-vivo. These comprised full, unbiased agonists, partial, G-protein biased agonists as well as antagonists for KOPr. In a broad in-vitro screening on binding affinities and activation of G-protein coupled receptor pathways HS666 was identified as partial, G-protein biased KOPr agonist. This was investigated in more detail in-vivo, testing anti-convulsant effects in the acute seizure model of pentylenetetrazole infusion in mice. Intraperitoneal administration of HS666 showed dose-dependent (0.3-10 mg/kg) and significant increase in the threshold for PTZ-induced seizures and reduced paroxysmal activity in the KA model. The central site of action and specific KOPr-mediated anti-seizure/anti-convulsant actions of HS666 were demonstrated with the selective KOPr antagonists, 5-guanidinonaltrindole and nor-binaltorphimine. Moreover, HS666 did not induce aversion or affected locomotor activity at therapeutic doses as tested by the conditioned place preference and locomotor activity tests. These findings indicate that HS666 has the prerequisite pharmacological characteristics of an effective drug in experimental epilepsy, by activating central KOPr to produce antiseizure/anticonvulsant effects and its reduced liability for adverse effects. Besides this, we could determine the role of the mTOR pathway in aversive effects of KOPr activation. This was achieved applying phosphoproteomic studies on distinct brain regions of mice treated with KOPr agonists inducing or not-inducing aversive effects. Our data give a much more in depth view of differences in pathway activation of KOR agonists inducing aversion compared to such not inducing aversion. In a side project we also investigated the anti-seizure effects of novel bumetanide derivatives with reduced diuretic effects and improved blood-brain-barrier penetration. Our data demonstrate the successful optimization of the pharmacological profile of bumetanide and the potential of the improved derivative BUM97 for the treatment of therapy-resistant TLE, in particular in combinatorial drug regimens with the GABA mimetic phenobarbital. Moreover we started to characterize the intrahippocampal kainic acid model in female mice.