Periodic Reporting for period 1 - PhotoTherEpi (Targeting epilepsy with phototherapeutics)
Periodo di rendicontazione: 2022-09-01 al 2025-02-28
As photo-activatable drugs (PDs) can be precisely controlled in space and time, caged and switchable photoactivatable drugs (CPDs, SPDs) are rapidly emerging as potential therapeutics for varied forms of cancer, vision loss, diabetes, or pain disorders. Despite their potential, PDs have not been exploited for epilepsy, a common, often debilitating neurological disorder. As 30% of epilepsies are medically intractable, and antiepileptic drugs often cause multi-organ side effects, PDs could break new therapeutic ground. PDs can be applied on demand, and locally activated/inactivated in single or multiple epileptic brain areas in a targeted fashion. This minimizes systemic side effects, and allows the application of potent drugs from other fields yet unthinkable in routine epileptology (e.g. general anesthetics). Importantly, being small molecules, different PDs can be combined or easily exchanged, and do not require protein expression. Using current and new PDs, we aim to control epileptic networks in vivo in a realistic epilepsy mouse model, and resected human brain tissue from patients with intractable epilepsy. Aim 1 will quantify antiepileptic potency of a range of PDs in human tissue using field potential and patch-clamp recordings, and cellular scale 2-photon imaging. In aim 2, PDs will be evaluated in vivo using wireless video-EEG, imaging and light-fiber-targeted drug photoactivation in chronically epileptic mice. PhotoTherEpi will establish targeted photopharmacology as a versatile, and powerful new approach to control hard-to-treat focal epilepsy. The approach could obviate the need for resective surgery in many cases, and be used in multi-focal epilepsy. Importantly, it may be clinically tested in the foreseeable future.
Activities:
Aim 1: Photocontrol of chronically epileptic human brain tissue following resective surgery
Mid-project, we have made major advances with regards to aim1. First, we have established two electrophysiology frameworks with cellular and field potential recordings, in combination with fiber-based light-activation. By now, we have completed the electrophysiological evaluation of 3 photodrug candidates listed in the grant agreement (GA), including testing in post-resective human brain tissue from patients with refractory epilepsy. These are patients we hope will primarily benefit from our research, as it may obviate the need for surgical resection in a substantial fraction of cases. Further, we have achieved the design and synthesis of two more antiepileptic phototherapeutics.
Specific activities/achievements:
1) A main effort in the last years lay on the development of different versions of the photodrugs listed in the GA. The versions have been evolved with regards to bioavailability (e.g. will the medication reach the brain?), balance between activatability and stability (e.g. compound should not be activatable by ambient environmental light, but should also not require light intensities that are not tolerated by biological tissue), and light activation wavelength.
2) In acute postresective brain tissue: basic evaluation of two previously published ion-channel-blockers that have not been exploited for epilepsy research, and one newly developed light-activatable drug as per GA. All three tested photodrugs could reliably block neuronal activity in vitro, while the new photodrug that has been developed within this ERC project appears to be most suitable for treatment of refractory human epilepsy, as it currently stands.
3) In cell culture systems: basic evaluation of two more light-activatable drugs in HEK293-cell culture patch-clamp recordings. Further, successful evaluation of one of these two compounds in multi-electrode cell culture recordings of synchronous network events.
4) First manuscript on photoactivatable drugs targeting epileptic networks in human brain tissue is now in preparation.
Aim 2: Targeting epileptogenesis and chronic focal epilepsy in vivo using phototherapeutics in mice
While most of the sub-aims of aim2 are subject to work during the second half of the project period, we have already made significant progress on several sub-aims of aim2 so far.
1) As described just above, we have developed several photoactivatable drugs towards applicability in intact bio-organisms, with a focus on photo-activatability, bio-availability, and tissue-light-tolerance. These steps are necessary prior to further evaluation of photoactivatable drugs in the intact organism.
2) We have evolved our chronic multimodal in vivo epilepsy models, which are now ideally suited to test photoactivatable drugs in the context of epileptic network activity in vivo. In this regard, two peer-reviewed publications have been achieved, and another one is in revision right now.
Aim 1: Photocontrol of chronically epileptic human brain tissue following resective surgery
Mid-project, we have made major advances with regards to aim1. First, we have established two electrophysiology frameworks with cellular and field potential recordings, in combination with fiber-based light-activation. By now, we have completed the electrophysiological evaluation of 3 photodrug candidates listed in the grant agreement (GA), including testing in post-resective human brain tissue from patients with refractory epilepsy. These are patients we hope will primarily benefit from our research, as it may obviate the need for surgical resection in a substantial fraction of cases. Further, we have achieved the design and synthesis of two more antiepileptic phototherapeutics.
Specific activities/achievements:
1) A main effort in the last years lay on the development of different versions of the photodrugs listed in the GA. The versions have been evolved with regards to bioavailability (e.g. will the medication reach the brain?), balance between activatability and stability (e.g. compound should not be activatable by ambient environmental light, but should also not require light intensities that are not tolerated by biological tissue), and light activation wavelength.
2) In acute postresective brain tissue: basic evaluation of two previously published ion-channel-blockers that have not been exploited for epilepsy research, and one newly developed light-activatable drug as per GA. All three tested photodrugs could reliably block neuronal activity in vitro, while the new photodrug that has been developed within this ERC project appears to be most suitable for treatment of refractory human epilepsy, as it currently stands.
3) In cell culture systems: basic evaluation of two more light-activatable drugs in HEK293-cell culture patch-clamp recordings. Further, successful evaluation of one of these two compounds in multi-electrode cell culture recordings of synchronous network events.
4) First manuscript on photoactivatable drugs targeting epileptic networks in human brain tissue is now in preparation.
Aim 2: Targeting epileptogenesis and chronic focal epilepsy in vivo using phototherapeutics in mice
While most of the sub-aims of aim2 are subject to work during the second half of the project period, we have already made significant progress on several sub-aims of aim2 so far.
1) As described just above, we have developed several photoactivatable drugs towards applicability in intact bio-organisms, with a focus on photo-activatability, bio-availability, and tissue-light-tolerance. These steps are necessary prior to further evaluation of photoactivatable drugs in the intact organism.
2) We have evolved our chronic multimodal in vivo epilepsy models, which are now ideally suited to test photoactivatable drugs in the context of epileptic network activity in vivo. In this regard, two peer-reviewed publications have been achieved, and another one is in revision right now.
In the epilepsy model of this project, we have recently unexpectedly revealed, while we were studying seizures, region-specific vulnerabilities for large depolarization waves that ride on seizures (Mitlasóczki et al, bioRxiv, 2024). We find that they are associated with specific, partly life-threatening symptoms known in clinical epileptology (e.g. post-ictal wandering). To some extent, we also recapitulated our results in refractory human focal epilepsy. To date, a similar mechanism had been described for sudden unexpected death in epilepsy patients (SUDEP), where seizure-associated spreading depolarization in the brain stem may be responsible for the failure of basic brain functions necessary for survival (e.g. breathing). Our findings point towards a more general role of this phenomenon in epilepsy. This could push reconsideration of the current EEG standard in the clinic, where the common 0.5 Hz high-pass filter has rendered these depolarization waves invisible for decades.