H. Steve White, Ph.D. - US grants

The specific aims of this research proposal are to determine the effects that the anticonvulsants phenytoin, carbamazepine, valproic acid, ethosuximide, acetazolamide, and diazepam have on cation and anion transport processes and intracellular pH homostasis of glial cells isolated from the cerebral cortices of 3-day old neonatal rats and maintained in isolated tissue culture. They include determination of the acute and chronic effects of each agent on: (1) the DNA and protein content of treated cultures, (2) the activity of the transport enzymes Na+/K+, Ca++/Mg++, and HCO3-ATPase, (3) the concentration of intracellular Na+, K+, and Cl, and ion flux measurements, (4) the activity of the glial specific enzyme carbonic anhydrase, (5) the membrane potential, resistance and conductance, (6) the intracellular pH, (7) the effect of specific inhibitors of cation and anion transport in the presence and absence of anticonvulsant drugs, (8) the effect of increasing extracellular K+, and (9) the effect of pentylenetetrazol alone and with each anticonvulsant agent. These studies will involve the disiplines of neuropharmacology, biochemistry, electrolyte and acid-base chemistry, eletrophysiology, cellular biology, and isolated tissue culture. The experiments are designed to test the hypothesis that changes in these parameters will enhance glial cell regulatory process thereby providing the CNS with an enhanced ability to regulate the extracellular fluid. Such experiments are important, for if a thorough understanding of the mechanisms of these drugs is to be obtained, a comprehensive examination of their effects on glial cells as well as neurons must be undertaken. When the mechanisms of these anticonvulsants are determined, new drugs exhibiting similar characteristics but with fewer side-effects can be made and tested for clinical efficacy.

The six specific aims of this competing renewal are designed to determine the effects of anticonvulsant and convulsant drugs on ion-transport processes, acid-base homeostasis and electrophysiology of astrocytes in tissue culture. These studies are designed to test the hypothesis that anticonvulsants in addition to their effects on neurons and synapses also stimulate astrocytic regulatory processes, and in so doing provide the CNS with an enhanced ability to regulate electrolyte, acid-base and neurotransmitter homeostasis within the CNS, thereby limiting seizure activity. Specifically, primary cultures of mouse cerebral cortical astrocytes will be acutely and chronically exposed to therapeutic concentrations of the prototype anticonvulsants phenytoin, carbamazepine, valproate, diazepam, ethosuximide, acetazolamide, and to the calcium channel blocker flunarizine. The effects of the above treatments on; the uptake, steady-state distribution and release of 22 Na+, 42 K+, 36 Cl- and 45 Ca++; the intracellular concentration of Na+, K+, Cl- and H+; the transport enzymes Na+/K+, Ca++/Mg++ -ATPase and the glial specific enzyme carbonic anhydrase; the electrophysiology (membrane potential, resistance and conductance); and the uptake of the neurotransmitter substance glutamate will be assessed. In addition, studies to characterize the effect of the receptor specific convulsants N-methyl-d-aspartate, and entylenetetrazol on the above parameters in untreated and anticonvulsant-treated (acute and chronic) astrocytes will also be conducted. The focus of this application does not differ significantly from that of the initial grant. Such an analysis is important for if a thorough understanding of the mechanisms of these drugs is to be obtained, a comprehensive examination of their effects on astrocytes as well as neurons must be undertaken. These studies will also enhance our understanding of the seizure process. Thus, through an increased understanding of the mechanisms of the currently available anticonvulsant drugs and the processes underlying seizure disorders, drugs with greater selectively and fewer adverse effects can be developed.

biomedical equipment purchase;

DESCRIPTION (provided by applicant): Despite recent advances in the treatment of epilepsy, more than 35-40% of the 2.5 million Americans receiving antiepileptic drugs (AED) remain refractory to existing AED therapies. In order to better understand the etiology of pharmacorestance to AED and to facilitate the development of innovative therapeutic approaches for the management of pharmacoresistant epilepsy, there is a great need to validate and systematically characterize drug effects in both in vivo and in vitro animal models that display persistent seizure activity (or seizure-like activity in vitro) which is refractory or responds poorly to at least two differentially acting AED at maximal tolerated doses. The goal of the current proposal is therefore to test the hypothesis that "the 'entorhinal-hippocampal slice' and the "lamotrigine (LTG)-resistant kindled rat' models are pharmacoresistant to established antiepileptic drugs and that AED found effective in one or both of these models would be novel relative to the currently available AED." This hypothesis will be tested by utilizing a two-tiered approach wherein the effects of multiple standard and investigational AED will be assessed according to the following two Specific Aims. Specific Aim 1 will evaluate the ability of a battery of mechanistically distinct established and investigational AED to eliminate spontaneous, electrographic seizure-like activity recorded in the medial entorhinal cortex in brain slices obtained from animals treated with kainic acid (KA). Specific Aim 2 will evaluate the ability of a battery of mechanistically distinct established and investigational AED to suppress focal and generalized seizures in LTG-resistant amygdala-kindled rats. Dose-response relationships will be determined in vitro for at least thirteen different "standard" and "investigational" AED therapies: phenytoin, carbamazepine, valproate, ethosuxamide, vigabatrin, topiramate, lamotrigine, felbamate, levetiracetam, harkoseride, tiagabine, valrocemide, and retigabine. Furthermore, these drugs will be evaluated in vivo for their ability to inhibit focal and generalized seizure activity in both the traditional and lamotrigine-resistant amygdala kindled rat models of temporal lobe epilepsy. These experiments will establish a comparative database for traditional and nontraditional AED in both in vitro and in vivo models, provide insight towards the predictive value of pharmacoresistance observed with in vitro screening paradigms for identifying pharmacoresistance observed with in vivo models of pharmacoresistant epilepsy, and set the stage for the development of future therapeutic interventions for the treatment of pharmacoresistant epilepsy.

DESCRIPTION (provided by applicant): Despite recent advances in the treatment of epilepsy, more than 35-40% of the 2.5 million Americans receiving antiepileptic drugs (AED) remain refractory to existing AED therapies. In order to better understand the etiology of pharmacorestance to AED and to facilitate the development of innovative therapeutic approaches for the management of pharmacoresistant epilepsy, there is a great need to validate and systematically characterize drug effects in both in vivo and in vitro animal models that display persistent seizure activity (or seizure-like activity in vitro) which is refractory or responds poorly to at least two differentially acting AED at maximal tolerated doses. The goal of the current proposal is therefore to test the hypothesis that "the 'entorhinal-hippocampal slice' and the "lamotrigine (LTG)-resistant kindled rat' models are pharmacoresistant to established antiepileptic drugs and that AED found effective in one or both of these models would be novel relative to the currently available AED." This hypothesis will be tested by utilizing a two-tiered approach wherein the effects of multiple standard and investigational AED will be assessed according to the following two Specific Aims. Specific Aim 1 will evaluate the ability of a battery of mechanistically distinct established and investigational AED to eliminate spontaneous, electrographic seizure-like activity recorded in the medial entorhinal cortex in brain slices obtained from animals treated with kainic acid (KA). Specific Aim 2 will evaluate the ability of a battery of mechanistically distinct established and investigational AED to suppress focal and generalized seizures in LTG-resistant amygdala-kindled rats. Dose-response relationships will be determined in vitro for at least thirteen different "standard" and "investigational" AED therapies: phenytoin, carbamazepine, valproate, ethosuxamide, vigabatrin, topiramate, lamotrigine, felbamate, levetiracetam, harkoseride, tiagabine, valrocemide, and retigabine. Furthermore, these drugs will be evaluated in vivo for their ability to inhibit focal and generalized seizure activity in both the traditional and lamotrigine-resistant amygdala kindled rat models of temporal lobe epilepsy. These experiments will establish a comparative database for traditional and nontraditional AED in both in vitro and in vivo models, provide insight towards the predictive value of pharmacoresistance observed with in vitro screening paradigms for identifying pharmacoresistance observed with in vivo models of pharmacoresistant epilepsy, and set the stage for the development of future therapeutic interventions for the treatment of pharmacoresistant epilepsy.

The primary goal of the modification to this contract is to discover small molecules that attenuate seizure activity precipitated by acute exposure to chemical threat agents defined as: toxic chemical agents that could be used in a terrorist attack against civilians, or those that could be released at toxic levels by accident or natural disaster. These include organophosphorus nerve agents and pesticides and cyanide. The further intent of modifying this contract is to take advantage of the economies of scale that already exist by utilizing a portion of the existing anti-convulsant screening mechanisms contractually already in place. We envision two tracts for newly submitted compounds. In the first, compounds will continue to be submitted to the program being screened under the current ASP anticonvulsant model at the rate of 750 per year. The second tract (the work described herein)will be directed specifically at uncovering potential leads against chemical threats. Several new models specific for these purposes are described below. In addition, relevant models already existing as part of the current ASP capacity will be employed to enhance the determination of pharmacological profiles in both CNS protection and toxicity of candidate compounds. It is expected that another 750 compounds can be evaluated in tract two. The combined capacity will be approximate 1500 compounds per year. Costs associated with the different tracts will be monitored and reported separately by the contractor. It is recognized that a combination of submission types will occur. These include: 1) submission to chemical threat screening for compounds that already have preliminary ASP data otherwise called transferred compounds 2) compounds submitted for dual evaluations (traditional ASP and chemical threats) and 3) compounds submitted solely for the tract two (chemical threat agents) portion. Tracking of expenditures for each type must be maintained and submitted in regular quarterly reports (see general requirement section).

DESCRIPTION (provided by applicant): The overall objective of this Translational Research Proposal is to synthesize, characterize, and advance an optimized galanin-based analog to IND filing so that a human proof-of-concept clinical trial with a novel first-in-class therapeutic can be conducted in patients with refractory epilepsy. Most of the available antiepileptic drugs (AEDs) exert their activity by modulating voltage- and/or receptor-gated ion channels. Despite treatment with currently available AEDs, approximately 25-40% of patients with refractory partial epilepsy continue to experience uncontrolled seizures, so clearly there is a need for a novel approach. The endogenous neuropeptide galanin and its associated receptors play an important role in the control of seizures and is therefore an attractive therapeutic target. However, developing neuropeptides as therapeutics has been challenging due to their intrinsic lack of metabolic stability and inability to cross the blood-brain-barrier. We have applied a novel technology platform to develop a galanin analog, NAX 5055, that is metabolically stable, maintains low nanomolar affinity for galanin receptors and following systemic administration is effective in an animal seizure model that displays resistance to the majority of first-line AEDs. The Aims outlined in this proposal are designed to optimize the NAX 5055 backbone and to advance one compound to an IND filing as follows: 1) identify one to six optimized galanin receptor 2-preferring analogs that display potent anticonvulsant activity and wide behavioral, cognitive and metabolic safety margins;2) select at least one IND candidate based on the most favorable pharmacological and ADME profiles;3) develop a formulation that maintains efficacy in animal models following systemic administration and is suitable for further preclinical and clinical development;4) and 5) perform all necessary IND-enabling preclinical studies. Successful completion of Aims 1 - 5 will allow the development of a clinical plan and submission of an IND application with the FDA to begin clinical trials of a new, first-in-class neurotherapy for refractory epilepsy. PUBLIC HEALTH RELEVANCE: Epilepsy affects over 50 million persons world-wide and approximately 25-40% of refractory partial epilepsy patients have uncontrolled seizures despite treatment with antiepileptic drugs. Galanin is an endogenous peptide in the brain that plays an important role in controlling seizures. The goal of this proposal is to apply our proprietary technology to develop a novel galanin-based therapy for treating this debilitating disorder.

DESCRIPTION (provided by applicant): Viral infections of the central nervous system (CNS) are associated with an increased risk for seizures, status epilepticus (SE), and the development of chronic epilepsy. We recently described a novel animal model of viral-induced epilepsy. Mice (C57BL/6) who receive intracerebral injections of Theiler's Murine Encephalomyelitis Virus (TMEV) display acute spontaneous seizures several days after infection, survive the initial infection and go on to develop spontaneous recurrent seizures. Preliminary data using C57BL/6 mice with various cytokines or cytokine receptors knocked-out have shown that these genetic manipulations can significantly alter the pathologic sequelae observed following TMEV injection of wildtype mice. Therefore, we propose to test our overall hypothesis that TMEV infection is associated with increased expression of TNF-a that contributes to increased neuronal excitability, acute seizures, neuropathology, and ultimately, epilepsy. The proposed experiments will lead to a greater understanding of the role of viral and immune contributions to acute seizures, altered neuronal and glial function, and epileptogenesis. We will use a multidisciplinary approach;including, chronic video-EEG monitoring and brain slice electrophysiology to: 1) test the hypothesis that the TNF-a pathway plays an important role in the development of chronic seizures following TMEV infection, and 2) test the hypothesis that TNF-a signaling mediates changes in synaptic and/or glial function in the hippocampus in TMEV infected mice. We anticipate that the results generated will provide a novel model in which to study the role of infection in the development of epilepsy. In addition, these experiments will provide important new insight into the role of TNF-a in cell death, synaptic transmission and epileptogenesis and set the stage for the development of novel therapeutic interventions for the prevention of infection-induced epilepsy. PUBLIC HEALTH RELEVANCE: Viral infections of the central nervous system are associated with an increased risk for seizures, status epilepticus, and the development of chronic epilepsy. We have developed a novel mouse model of viral induced epilepsy using cortical injections of Theiler's Murine Encephalitis Virus (TMEV) to study epileptogenesis. The proposed multidisciplinary set of experiments will lead to a greater understanding of the role of viral and host immune contributions to acute seizures, altered neuronal function, and epileptogenesis.