Ex Vivo and In Vivo Studies of the Lysophospholipids Edelfosine and Mitelfosine to Develop Novel Anti‐Epileptic Therapies

Abstract

Epilepsy is a chronic neurological disorder characterized by repeated seizures. Today, most anti‐epileptic therapies are a combination of anticonvulsant drugs that work on the excitability of neurons by inhibiting sodium and calcium channels, enhancing GABAergic transmission, inhibiting glutamate transmission or have several combined actions. However, these drugs have detrimental side effects, such as congenital disabilities and psychiatric comorbidities such as mood disorders, depression, and anxiety. In addition, ~35% of adults and ~15% of children epilepsy patients are resistant to these available therapies. Thus, it is critical to study new drugs against epileptic seizures that target neuronal mechanisms different from those used by classical anticonvulsants. Edelfosine (Ef) is a lysophospholipid that works as a phospholipase‐C‐β3 (PLCβ3) inhibitor. PLCβ3 is an effector of the G protein coupled to muscarinic acetylcholine receptors that produces inositol triphosphate and diacylglycerol increasing the release of calcium (Ca+2) from the calcium‐storing organelles. This cytoplasmic Ca2+ regulates several signaling cascades including opening of ion channels that leads neurons to excitotoxic death. Thus, Ef utilizes a neuroprotective mechanism against excitotoxicity that does not act directly on ion channels. This makes Ef a superior drug against muscarinic hyperstimulation. Unfortunately, Ef is not effective within the brain due to the blood‐brain barrier's (BBB) high selectivity, which does not allow its entry. Our project aims to deliver therapeutic concentrations of Ef to the brain to diminish neuronal excitotoxicity. Our first studies test the ex vivo effect of Ef in rat hippocampal slices after two different muscarinic hyperstimulation (excitotoxic insults). Our results showed that Ef could decrease brain excitotoxicity from both insults by inhibiting the PLCβ3 enzyme. This ex vivo preliminary data demonstrates for the first time in a brain model that exposure of hippocampal slices to Ef induced a significant recovery after a muscarinic hyperstimulation insult. For the next studies, we developed two non‐BBB‐targeted serum albumin (SA)‐based drug delivery system (DDS) loading Ef and the Ef analog, mitelfosine (Mf). We characterized the particle physical properties of these Ef‐SA DDS and Mf‐SA DDS using dynamic light scattering and circular dichroism. For both DDS, SA showed characteristic structural changes compared to the native SA, demonstrating the capacity of SA to load these two lysophospholipids. We also performed a short in vivo experiment for four days administrating these DDS to determine their safety in mice. From these in vivo preliminary results, we concluded that Ef‐SA DDS and Mf‐SA DDS showed no sign of toxicity and did not affect the weight, grooming or mobility. Future experiments will focus on the development of BBB‐targeted Ef‐SA DDS and Mf‐SA DDS and their in vivo effect in an epilepsy mouse model.