Search

Objective Infection with Theiler's Murine Encephalomyelitis Virus (TMEV) in C57Bl/6J mice results in handling-induced seizures and is useful for evaluating compounds effective against infection-induced seizures. However, to date only a few compounds have been evaluated in this model, and a comprehensive study of antiseizure medications (ASMs) has not yet been performed. Furthermore, as the TMEV infection produces marked neuroinflammation, an evaluation of prototype anti-inflammatory compounds is needed as well. Methods Male C57Bl/6J mice were inoculated with TMEV (day 0) followed by daily administrations of test compounds (day 3-7) and subsequent handling sessions (day 3-7). Doses of ASMs, comprising several mechanistic classes, were selected based on previously published data demonstrating the effect of these compounds in reducing seizures in the 6 Hz model of pharmacoresistant seizures. Doses of anti-inflammatory compounds, comprising several mechanistic classes, were selected based on published evidence of reduction of inflammation or inflammation-related endpoints. Results Several prototype ASMs reduced acute seizures following TMEV infection: lacosamide, phenytoin, ezogabine, phenobarbital, tiagabine, gabapentin, levetiracetam, topiramate, and sodium valproate. Of these, phenobarbital and sodium valproate had the greatest effect (>95% seizure burden reduction). Prototype anti-inflammatory drugs celecoxib, dexamethasone, and prednisone also moderately reduced seizure burden. Significance The TMEV model is utilized by the Epilepsy Therapy Screening Program (ETSP) as a tool for evaluation of novel compounds. Compounds reducing seizures in the TMEV comprise distinct mechanistic classes, some with mechanisms of action that extend beyond traditional ASMs.

The discovery and development of novel antiseizure drugs (ASDs) that are effective in controlling pharmacoresistant spontaneous recurrent seizures (SRSs) continues to represent a significant unmet clinical need. The Epilepsy Therapy Screening Program (ETSP) has undertaken efforts to address this need by adopting animal models that represent the salient features of human pharmacoresistant epilepsy and employing these models for preclinical testing of investigational ASDs. One such model that has garnered increased interest in recent years is the mouse variant of the Intra-Amygdala Kainate (IAK) microinjection model of mesial temporal lobe epilepsy (MTLE). In establishing a version of this model, several methodological variables were evaluated for their effect(s) on pertinent quantitative endpoints. Although administration of a benzodiazepine 40 minutes after kainate (KA) induced status epilepticus (SE) is commonly used to improve survival, data presented here demonstrates similar outcomes (mortality, hippocampal damage, latency periods, and 90-day SRS natural history) between mice given midazolam and those that were not. Using a version of this model that did not interrupt SE with a benzodiazepine, a 90-day natural history study was performed and survival, latency periods, SRS frequencies and durations, and SRS clustering data were quantified. Finally, an important step towards model adoption is to assess the sensitivities or resistances of SRSs to a panel of approved and clinically used ASDs. Accordingly, the following ASDs were evaluated for their effects on SRSs in these mice: phenytoin (20 mg/kg, b.i.d.), carbamazepine (30 mg/kg, t.i.d.), valproate (240 mg/kg, t.i.d.), diazepam (4 mg/kg, b.i.d.), and phenobarbital (25 and 50 mg/kg, b.i.d.). Valproate, diazepam, and phenobarbital significantly attenuated SRS frequency relative to vehicle controls at doses devoid of observable adverse behavioral effects. Only diazepam significantly increased seizure freedom. Neither phenytoin nor carbamazepine significantly altered SRS frequency or freedom under these experimental conditions. These data demonstrate that SRSs in this IAK model of MTLE are pharmacoresistant to two representative sodium channel-inhibiting ASDs (phenytoin and carbamazepine) but not to GABA receptor modulating ASDs (diazepam and phenobarbital) or a mixed-mechanism ASD (valproate). Accordingly, this model is being incorporated into the NINDS-funded ETSP testing platform for treatment resistant epilepsy.HighlightsAn intra-amygdala kainate model of TLE was evaluated for pharmacoresistant seizuresAdministration of midazolam during status epilepticus did not affect mortalityModel characteristics were evaluated over a 90-day natural history studySpontaneous seizures were resistant to phenytoin and carbamazepineSpontaneous seizures were sensitive to valproic acid, diazepam, and phenobarbital

SummaryObjectiveDravet syndrome (DS) is a rare, but catastrophic genetic epilepsy, with 80% of patients with carrying a mutation in the SCN1A gene. Currently, no anti-seizure drug (ASD) exists that adequately controls seizures. Patients with DS often present clinically with a febrile seizure and generalized tonic-clonic seizures that continue throughout life. To facilitate the development of ASDs for DS, the contract site of the NINDS Epilepsy Therapy Screening Program (ETSP) has evaluated a mouse model of DS using the conditional knock-in Scn1aA1783V/WT mouse.MethodsSurvival rates and temperature thresholds for Scn1aA1783V/WT were determined. Prototype ASDs were administered via intraperitoneal injections at the time-to-peak effect, which was previously determined, prior to the induction of hyperthermia-induced seizures. Protection was determined if ASDs significantly increased the temperature at which Scn1aA1783V/WT mice seized.ResultsApproximately 50% of Scn1aA1783V/WT survive to adulthood and all have hyperthermia-induce seizures. The results suggest that hyperthermia-induced seizures in this model of DS are highly refractory to a battery of ASDs. Exceptions were clobazam, tiagabine, and the combination of clobazam and valproic acid with add-on stiripentol, which elevated seizure thresholdsSignificanceOverall, the data demonstrate the proposed model for DS is suitable for screening novel compounds for the ability to block hyperthermia-induced seizures and heterozygous mice can be evaluated repeatedly over the course of several weeks, allowing for higher throughput screening.Key PointsScn1aA1783V/WT mice have a 50% survival rate and all have hyperthermia-induced seizures.Common DS treatments such as CLB and combinatorial therapy of CLB, VPA, and STP increase temperature thresholds in Scn1aA1783V/WT mice.Sodium channel blockers, such as CBZ and LTG, decrease temperature thresholds of Scn1aA1783V/WT mice as predicted.Scn1aA1783V/WT mice are highly pharmacoresitant to common ASDsThe Scn1aA1783V/WT may be a useful preclinical drug screening platform for the treatment of DS.

Objectives Cannabidiolic acid (CBDa) is pharmacologically unique from cannabidiol (CBD), but its chemical instability poses challenges for potential clinical utility. Here, we used magnesium ions to stabilize two cannabidiolic acid-enriched hemp extracts (Mg-CBDa and Chylobinoid, the latter of which also contains minor cannabinoid constituents) and compared their anticonvulsant activities with CBD in the maximal electroshock seizure test (MES) in rats. Methods Sprague-Dawley rats received intraperitoneal (i.p.) injections of Chylobinoid, Mg-CBDa, or CBD at varying doses at discrete time points. Rats were challenged with a 0.2 s, 60 Hz, 150 mA corneal stimulation and evaluated for resultant hindlimb tonic extension. Dose-response relationships were calculated using Probit analysis and statistical significance was assessed with a two-sample z-test. Results Median effective doses (ED50) and 95% confidence intervals were calculated for each compound and adjusted according to percentage of CBDa (w/w): Chylobinoid: 76.7 (51.7 - 109.2) mg/kg. Mg-CBDa: 115.4 (98.8 - 140.9) mg/kg. CBD: 68.8 (56.6 - 80.0) mg/kg. Significance CBDa-enriched hemp extracts exhibited dose-dependent protection in the MES model at doses comparable, but not more effective than, CBD. Chylobinoid was more effective than Mg-CBDa despite lower CBDa content. Test compounds should be compared by sub-chronic dosing in the MES test in order to assess safety and pharmacokinetic profiles. CBDa should be evaluated in pharmacoresistant and chronic animal models of epilepsy.

Objective Approximately 30% of patients with epilepsy are refractory to existing antiseizure drugs (ASDs). Given that the properties of the central nervous systems of these patients are likely to be altered due to their epilepsy, tissues from rodents that have undergone epileptogenesis might provide a therapeutically relevant disease substrate for identifying compounds capable of attenuating pharmacoresistant seizures. To facilitate the development of such a model, this study describes the effects of classical glutamate receptor antagonists and 20 ASDs on recurrent epileptiform discharges (REDs) in brain slices derived from the kainate-induced status epilepticus model of temporal lobe epilepsy (KA-rats). Methods Horizontal brain slices containing the medial entorhinal cortex (mEC) were prepared from KA-rats, and REDs were recorded from the superficial layers. 6-cyano-7-nitroquinoxaline-2,3-dione, (2R)-amino-5-phosphonovaleric acid, tetrodotoxin, or ASDs were bath applied for 20 minutes. Concentration-dependent effects and half maximal effective concentration values were determined for RED duration, frequency, and amplitude. Results ASDs targeting sodium and potassium channels (carbamazepine, eslicarbazepine, ezogabine, lamotrigine, lacosamide, phenytoin, and rufinamide) attenuated REDs at concentrations near their average therapeutic plasma concentrations. γ-aminobutyric acid (GABA)ergic synaptic transmission-modulating ASDs (clobazam, midazolam, phenobarbital, stiripentol, tiagabine, and vigabatrin) attenuated REDs only at higher concentrations and, in some cases, prolonged RED durations. ASDs with other/mixed mechanisms of action (bumetanide, ethosuximide, felbamate, gabapentin, levetiracetam, topiramate, and valproate) and glutamate receptor antagonists weakly or incompletely inhibited RED frequency, increased RED duration, or had no significant effects. Significance Taken together, these data suggest that epileptiform activity recorded from the superficial layers of the mEC in slices obtained from KA-rats is differentially sensitive to existing ASDs. The different sensitivities of REDs to these ASDs may reflect persistent molecular, cellular, and/or network-level changes resulting from disease. These data are expected to serve as a foundation upon which future therapeutics may be differentiated and assessed for potentially translatable efficacy in patients with refractory epilepsy.

The series of experiments herein evaluated prototype drugs representing different mechanisms of antiseizure, antinociceptive or antidepressant action in a battery of preclinical pain models in adult male CF#1 mice (formalin, writhing, and tail flick) and Sprague Dawley rats partial sciatic nerve ligation (PSNL). In the formalin assay, phenytoin (PHT, 6 mg/kg), sodium valproate (VPA, 300 mg/kg), amitriptyline (AMI, 7.5 and 15 mg/kg), gabapentin (GBP, 30 and 70 mg/kg), tiagabine (TGB, 5 and 15 mg/kg), and acetominophen (APAP, 250 and 500 mg/kg) reduced both phases of the formalin response to ≤ 25% of vehicle-treated mice. In the acetic acid induced writhing assay, VPA (300 mg/kg), ethosuximide (ETX, 300 mg/kg), morphine (MOR, 5 & 10 mg/kg), GBP (10, 30, and 60 mg/kg), TGB (15 mg/kg), levetiracetam (LEV, 300 mg/kg), felbamate (FBM, 80 mg/kg) and APAP (250 mg/kg) reduced writhing to ≤ 25% of vehicle-treated mice. In the tail flick test, MOR (1.25-5 mg/kg), AMI (15 mg/kg) and TGB (5 mg/kg) demonstrated significant antinociceptive effects. Finally, carbamazepine (CBZ, 20 and 50 mg/kg), VPA, MOR (2 and 4 mg/kg), AMI (12 mg/kg), TPM (100 mg/kg), lamotrigine (LTG, 40 mg/kg), GBP (60 mg/kg), TGB (15 mg/kg), FBM (35 mg/kg), and APAP (250 mg/kg) were effective in the PSNL model. Thus, TGB was the only prototype compound with significant analgesic effects in each of the four models, while AMI, GBP, APAP, and MOR each improved three of the four pain phenotypes. This study highlights the importance evaluating novel targets in a variety of pain phenotypes.

SummaryObjective The mouse 6 Hz model of psychomotor seizures is a well-established and commonly used preclinical model for antiseizure drug (ASD) discovery. Despite its widespread use both in the identification and differentiation of novel ASDs in mice, a corresponding assay in rats has not been developed. We established a method for 6 Hz seizure induction in rats, with seizure behaviors similar to those observed in mice including head nod, jaw clonus, and forelimb clonus. Methods A convulsive current that elicits these seizure behaviors in 97% of rats (CC97) was determined using a Probit analysis. Numerous prototype ASDs were evaluated in this model using stimulus intensities of 1.5× and 2× the CC97, which is comparable to the approach used in the mouse 6 Hz seizure model (e.g., 32 and 44 mA stimulus intensities). The ASDs evaluated include carbamazepine, clobazam, clonazepam, eslicarbazepine, ethosuximide, ezogabine, gabapentin, lacosamide, lamotrigine, levetiracetam, phenobarbital, phenytoin, rufinamide, tiagabine, topiramate, and sodium valproate. Median effective dose (ED50) and median toxic (motor impairment) dose (TD50) values were obtained for each compound. Results Compounds that were effective at the 1.5 × CC97 stimulus intensity at protective index (PI) values >1 included clobazam, ethosuximide, ezogabine, levetiracetam, phenobarbital, and sodium valproate. Compounds that were effective at the 2 × CC97 stimulus intensity at PI values >1 included ezogabine, phenobarbital, and sodium valproate. Significance In a manner similar to the use of the mouse 6 Hz model, development of a rat 6 Hz test will aid in the differentiation of ASDs, as well as in study design and dose selection for chronic rat models of pharmacoresistant epilepsy. The limited number of established ASDs with demonstrable efficacy at the higher stimulus intensity suggests that, like the mouse 6 Hz 44 mA model, the rat 6 Hz seizure model may be a useful screening tool for pharmacoresistant seizures.

The potential clinical utility of galanin peptidic analogs has been hindered by poor metabolic stability, lack of brain penetration, and hyperglycemia. In addition to possessing potent anticonvulsant efficacy, galanin analogs are analgesic in various assays. The purpose of these studies was to evaluate the lead galanin receptor type 2 (GalR2)-preferring analog, NAX 810-2, in various pain assays, as well as determine any potential for insulin inhibition, growth hormone stimulation, and cognitive impairment. NAX 810-2 was evaluated in mouse (carrageenan, formalin, tail flick, plantar incision) and rat pain models (partial sciatic nerve ligation). NAX 810-2 dose-dependently increased paw withdrawal latency following plantar administration of carrageenan (ED50 4.7 mg/kg). At a dose of 8 mg/kg, NAX 810-2 significantly attenuated nociceptive behaviors following plantar administration of formalin, and this was observed for both phase I (acute) and phase II (inflammatory) components of the formalin behavioral response. NAX-810-2 was active at higher doses in the mouse tail flick model (ED50 20.2 mg/kg) and similarly, reduced mechanical allodynia following plantar incision in mice at a dose of 24 mg/kg. NAX 810-2 also reduced mechanical allodynia in the partial sciatic nerve ligation model at a dose of 4 mg/kg. In addition, NAX 810-2 did not impair insulin secretion at doses of 2.5 and 8 mg/kg (acutely) or at a dose of 8 mg/kg given daily for 5 days. Similarly, 8 mg/kg (twice daily, 5 days) of NAX 810-2 did not increase growth hormone levels. These results demonstrate that NAX 810-2 possesses a favorable pre-clinical profile as a novel and first-in-class analgesic.

Objective C57BL/6J mice infected with Theiler's murine encephalomyelitis virus (TMEV) develop acute behavioral seizures in the first week of infection and later develop chronic epilepsy. The TMEV model provides a useful platform to test novel antiseizure therapeutics. The present study was designed to test the efficacy of cannabidiol (CBD) in reducing acute seizures induced by viral infection. Methods C57BL/6J mice were infected intracortically with 2 × 105 plaque‐forming units of TMEV. Mice were divided into two treatment groups—1) CBD‐treated mice and 2) vehicle‐treated mice. Frequency and severity of acute seizures were evaluated by video‐monitoring the mice four times daily by the experimenter blinded to the treatment group. Results Cannabidiol (180 mg/kg; 360 mg/kg/day) decreased both the frequency and severity of acute behavioral seizures following TMEV infection, but 150 mg/kg of CBD did not improve overall seizure outcome. The time to peak effect (TPE) of CBD in the 6 Hz 32 mA psychomotor seizure test using C57BL/6J mice was observed at 2 hours post‐CBD treatment. Interestingly, CBD (150 mg/kg) significantly reduced frequency and severity of TMEV‐induced acute seizures at 2 hours post‐CBD treatment. These results suggest that CBD could be effective in decreasing TMEV‐induced acute seizures when the seizure test is conducted at the TPE of CBD. Significance Cannabinoids are increasingly studied for their potential antiseizure effects. Several preclinical and clinical studies provide evidence that CBD could be an effective therapy for intractable epilepsies. The present study corroborates those previous findings and provides an opportunity to investigate pharmacokinetics, pharmacodynamics, and mechanism(s) of antiseizure effects of CBD in the TMEV model, which may help to design future clinical studies more effectively.

Digital therapeutics (software as a medical device) and mobile health (mHealth) technologies offer a means to deliver behavioral, psychosocial, disease self-management and music-based interventions to improve therapy outcomes for chronic diseases, including pain and epilepsy. To explore new translational opportunities in developing digital therapeutics for neurological disorders, and their integration with pharmacotherapies, we examined analgesic and antiseizure effects of specific musical compositions in mouse models of pain and epilepsy. The music playlist was created based on the modular progression of Mozart compositions for which reduction of seizures and epileptiform discharges were previously reported in people with epilepsy. Our results indicated that music-treated mice exhibited significant analgesia and reduction of paw edema in the carrageenan model of inflammatory pain. Among analgesic drugs tested (ibuprofen, cannabidiol (CBD), levetiracetam, and the galanin analog NAX 5055), music intervention significantly decreased paw withdrawal latency difference in ibuprofen-treated mice and reduced paw edema in combination with CBD or NAX 5055. To the best of our knowledge, this is the first animal study on music-enhanced antinociceptive activity of analgesic drugs. In the plantar incision model of surgical pain, music-pretreated mice had significant reduction of mechanical allodynia. In the corneal kindling model of epilepsy, the cumulative seizure burden following kindling acquisition was lower in animals exposed to music. The music-treated group also exhibited significantly improved survival, warranting further research on music interventions for preventing Sudden Unexpected Death in Epilepsy (SUDEP). We propose a working model of how musical elements such as rhythm, sequences, phrases and punctuation found in K.448 and K.545 may exert responses via parasympathetic nervous system and the hypothalamic-pituitary-adrenal (HPA) axis. Based on our findings, we discuss: (1) how enriched environment (EE) can serve as a preclinical surrogate for testing combinations of non-pharmacological modalities and drugs for the treatment of pain and other chronic diseases, and (2) a new paradigm for preclinical and clinical development of therapies leading to drug-device combination products for neurological disorders, depression and cancer. In summary, our present results encourage translational research on integrating non-pharmacological and pharmacological interventions for pain and epilepsy using digital therapeutics.

Objective We previously demonstrated that positive allosteric modulators (PAMs) of metabotropic glutamate subtype 2 (mGlu2 ) receptors have potential synergistic interactions with the antiseizure drug levetiracetam (LEV). The present study utilizes isobolographic analysis to evaluate the combined administration of JNJ-46356479, a selective and potent mGlu2 PAM, with LEV as well as sodium valproate (VPA) and lamotrigine (LTG). Methods The anticonvulsant efficacy of JNJ-46356479 was evaluated in the 6-Hz model of psychomotor seizures in mice. JNJ-46356479 was administered in combination with LEV using 3 fixed dose-ratio treatment groups in the mouse 6-Hz (44-mA) seizure test. The combination of JNJ-46356479 with LEV was also evaluated in the mouse corneal kindling model. The potential interactions of JNJ-46356479 with the antiseizure drugs VPA and LTG were also evaluated using fixed dose-ratio combinations. Plasma levels were obtained for analysis of potential pharmacokinetic interactions for each combination studied in the mouse 6-Hz model. Results JNJ-46356479 was active in the 6-Hz model at both 32-mA and 44-mA stimulus intensities (median effective dose = 2.8 and 10.2 mg/kg, respectively). Using 1:1, 1:3, and 3:1 fixed dose-ratio combinations (LEV:JNJ-46356479), coadministration was significantly more potent than predicted for additive effects, and plasma levels suggest this synergism was not due to pharmacokinetic interactions. Studies in kindled mice further demonstrate the positive pharmacodynamic interaction of LEV with JNJ-46356479. Using 1:1 dose-ratio combinations of JNJ-46356479 with either VPA or LTG, there were no significant differences observed for coadministration. Significance These studies demonstrate a synergistic interaction of JNJ-46356479 with LEV, whereas no such effect occurred for JNJ-46356479 with either VPA or LTG. The synergy seems therefore to be specific to LEV, and the combination LEV/mGlu2 PAM has the potential to result in a rational polypharmacy approach to treat patients with refractory epilepsy, once it has been confirmed in clinical studies.

Cannabidiol (CBD) is a cannabinoid component of marijuana that has no significant activity at cannabinoid receptors or psychoactive effects. There is considerable interest in CBD as a therapy for epilepsy. Almost a third of epilepsy patients are not adequately controlled by clinically available anti-seizure drugs (ASDs). Initial studies appear to demonstrate that CBD preparations may be a useful treatment for pharmacoresistant epilepsy. The National Institute of Neurological Disorders and Stroke (NINDS) funded Epilepsy Therapy Screening Program (ETSP) investigated CBD in a battery of seizure models using a refocused screening protocol aimed at identifying pharmacotherapies to address the unmet need in pharmacoresistant epilepsy. Applying this new screening workflow, CBD was investigated in mouse 6 Hz 44 mA, maximal electroshock (MES), corneal kindling models and rat MES and lamotrigine-resistant amygdala kindling models. Following intraperitoneal (i.p.) pretreatment, CBD produced dose-dependent protection in the acute seizure models; mouse 6 Hz 44 mA (ED50 164 mg/kg), mouse MES (ED50 83.5 mg/kg) and rat MES (ED50 88.9 mg/kg). In chronic models, CBD produced dose-dependent protection in the corneal kindled mouse (ED50 119 mg/kg) but CBD (up to 300 mg/kg) was not protective in the lamotrigine-resistant amygdala kindled rat. Motor impairment assessed in conjunction with the acute seizure models showed that CBD exerted seizure protection at non-impairing doses. The ETSP investigation demonstrates that CBD exhibits anti-seizure properties in acute seizure models and the corneal kindled mouse. However, further preclinical and clinical studies are needed to determine the potential for CBD to address the unmet needs in pharmacoresistant epilepsy.

The successful identification of promising investigational therapies for the treatment of epilepsy can be credited to the use of numerous animal models of seizure and epilepsy for over 80 years. In this time, the maximal electroshock test in mice and rats, the subcutaneous pentylenetetrazol test in mice and rats, and more recently the 6 Hz assay in mice, have been utilized as primary models of electrically or chemically-evoked seizures in neurologically intact rodents. In addition, rodent kindling models, in which chronic network hyperexcitability has developed, have been used to identify new agents. It is clear that this traditional screening approach has greatly expanded the number of marketed drugs available to manage the symptomatic seizures associated with epilepsy. In spite of the numerous antiseizure drugs (ASDs) on the market today, the fact remains that nearly 30% of patients are resistant to these currently available medications. To address this unmet medical need, the National Institute of Neurological Disorders and Stroke (NINDS) Epilepsy Therapy Screening Program (ETSP) revised its approach to the early evaluation of investigational agents for the treatment of epilepsy in 2015 to include a focus on preclinical approaches to model pharmacoresistant seizures. This present report highlights the in vivo and in vitro findings associated with the initial pharmacological validation of this testing approach using a number of mechanistically diverse, commercially available antiseizure drugs, as well as several probe compounds that are of potential mechanistic interest to the clinical management of epilepsy.

Hydrocarbon stapling is an effective strategy to stabilize the helical conformation of bioactive peptides. Here we describe application of stapling to anticonvulsant neuropeptides, galanin (GAL) and neuropeptide Y (NPY), that are implicated in modulating seizures in the brain. Dicarba bridges were rationally introduced into minimized analogs of GAL and NPY resulting in increased α-helical content, in vitro metabolic stability and n -octanol/water partitioning coefficient (log D ). The stapled analogs retained agonist activities towards their respective receptors and suppressed seizures in a mouse model of epilepsy.

Chronic pain is a multifactorial disease comprised of both inflammatory and neuropathic components that affect ∼20% of the world's population. sec-Butylpropylacetamide (SPD) is a novel amide analogue of valproic acid (VPA) previously shown to possess a broad spectrum of anticonvulsant activity. In this study, we defined the pharmacokinetic parameters of SPD in rat and mouse, and then evaluated its antinociceptive potential in neuropathic and acute inflammatory pain models. In the sciatic nerve ligation (SNL) model of neuropathic pain, SPD was equipotent to gabapentin and more potent than its parent compound VPA. SPD also showed either higher or equal potency to VPA in the formalin, carrageenan, and writhing tests of inflammatory pain. SPD showed no effects on compound action potential properties in a sciatic nerve preparation, suggesting that its mechanism of action is distinct from local anesthetics and membrane stabilizing drugs. SPD's activity in both neuropathic and inflammatory pain warrants its development as a potential broad-spectrum anti-nociceptive drug.

Using molecular phylogeny has accelerated the discovery of peptidic ligands targeted to ion channels and receptors. One clade of venomous cone snails, Asprella, appears to be significantly enriched in conantokins, antagonists of N-methyl d-aspartate receptors (NMDARs). Here, we describe the characterization of two novel conantokins from Conus rolani, including conantokin conRl-B that has shown an unprecedented selectivity for blocking NMDARs that contain NR2B subunits. ConRl-B shares only some sequence similarity with the most studied NR2B selective conantokin, conG. The divergence between conRl-B and conG in the second inter-Gla loop was used to design analogues for structure-activity studies; the presence of Pro10 was found to be key to the high potency of conRl-B for NR2B, whereas the e-amino group of Lys8 contributed to discrimination in blocking NR2B- and NR2A-containing NMDARs. In contrast to previous findings for Tyr5 substitutions in other conantokins, conRl-B[L5Y] showed potencies on the four NR2 NMDA receptor subtypes that were similar to those of the native conRl-B. When delivered into the brain, conRl-B was active in suppressing seizures in the model of epilepsy in mice, consistent with NR2B-containing NMDA receptors being potential targets for antiepileptic drugs. Circular dichroism experiments confirmed that the helical conformation of conRl-B is stabilized by divalent metal ions. Given the clinical applications of NMDA antagonists, conRl-B provides a potentially important pharmacological tool for understanding the differential roles of NMDA receptor subtypes in the nervous system. This work shows the effectiveness of coupling molecular phylogeny, chemical synthesis, and pharmacology for discovering new bioactive natural products.

The glycosylation of neuroactive peptides is a promising strategy to treat neurological and psychiatric disorders. Herein we investigated the effects of site-specific glycosylation of neurotensin (NT). The glycosylated analogues have low-nanomolar affinities and agonist activities toward NTS1, and suppress seizures with sub-picomolar potency. Our work points to a new research direction of exploring BBB-permeable NT analogues as potential first-in-class antiepileptic drugs. Neurotensin (NT) is an endogenous neuropeptide involved in a variety of central and peripheral neuromodulatory effects. Herein we show the effects of site-specific glycosylation on the in vitro and in vivo pharmacological properties of this neuropeptide. NT analogues containing O-linked disaccharides (β-melibiose and α-TF antigen) or β-lactose units linked by a PEG3 spacer were designed and chemically synthesized using Fmoc chemistry. For the latter analogue, Fmoc-Glu-(β-Lac-PEG3-amide) was prepared. Our results indicate that the addition of the disaccharides does not negatively affect the sub-nanomolar affinity or the low-nanomolar agonist potency for the neurotensin receptor subtype 1 (NTS1). Interestingly, three glycosylated analogues exhibited sub-picomolar potency in the 6 Hz limbic seizure mouse model of pharmacoresistant epilepsy following intracerebroventricular administration. Our results suggest for the first time that chemically modified NT analogues may lead to novel antiepileptic therapies.

Disruptions in GABAergic neurotransmission have been implicated in numerous CNS disorders, including epilepsy and neuropathic pain. Selective inhibition of neuronal and glial GABA transporter subtypes may offer unique therapeutic options for regaining balance between inhibitory and excitatory systems. The ability of two GABA transport inhibitors to modulate inhibitory tone via inhibition of mGAT1 (tiagabine) or mGAT2/BGT-1 (N-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]-4-(methylamino-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol), also known as EF1502) was evaluated using an in vitro model of spontaneous interictal-like bursting (SB). SBs were recorded extracellularly in combined mEC-HC horizontal brain slices (400 microm; 31+/-1 degrees C) obtained from KA-treated rats. Slice recordings demonstrated that EF1502 exhibited a concentration-dependent reduction in SB frequency. EF1502 significantly reduced SB rate to 32% of control at the 30 microM concentration, while reducing the area and duration of SB activity to 60% and 46% of control, respectively, at the 10 microM concentration. In contrast, the GAT1 selective inhibitor tiagabine (3, 10, and 30 microM) was unable to significantly reduce the frequency of SB activity in the mEC, despite significantly reducing both the duration (51% of control) and area (58% of control) of the SB at concentrations as low as 3 microM. The ability of EF1502, but not tiagabine, to inhibit SBs in the mEC suggests that this in vitro model of pharmacoresistant SB activity is useful to differentiate between novel anticonvulsants with similar mechanisms of action and suggests a therapeutic potential for non-GAT1 transport inhibitors.

Hyperexcitability in the medial entorhinal cortex-hippocampal (mEC-HC) circuit in the initial weeks after prolonged seizure activity may contribute to the epileptogenic process in animal models of temporal lobe epilepsy (TLE). The present study examined combined mEC-HC slices (400 microm) using field potential recordings 1-2 weeks following the multiple administration, low-dose kainic acid (KA) model of TLE [Hellier, J.L., Patrylo, P.R., Buckmaster, P.S., Dudek, F.E., 1998. Recurrent spontaneous motor seizures after repeated low-dose systemic treatment with kainate: assessment of a rat model of temporal lobe epilepsy. Epilepsy Res. 31, 73-84]. Field potential recordings in slices from KA-treated rats demonstrated hallmarks of hyperexcitability in the mEC and in the CA1 and CA3 cell body regions of the HC. Spontaneous burst (SB) activity was observed under baseline recording conditions in the mEC of several slices from KA-treated rats, but not in the slices from saline-treated control rats. Elevating ACSF [K(+)](o) (6mM) in the presence of picrotoxin (50 microM) increased SB rates in all slices tested. However, there was a significantly shorter latency to onset of bursting and prolonged evoked response durations in layer II of the mEC of slices from KA-treated rats versus those from controls. Neither carbamazepine (CBZ) nor phenytoin (PHT) abolished SB activity in slices from KA-treated rats; whereas, SB activity in slices from control rats was dose-dependently reduced at 100 microM CBZ. In contrast, the novel anticonvulsant retigabine (RGB) dramatically reduced SB frequency in both control and KA-treated groups. The hyperexcitability observed in combined mEC-HC brain slices from KA-treated rats suggests that the mEC, as well as the HC, may contribute to the epileptogenic process after KA-induced seizure activity. This model may provide an efficient, flexible in vitro paradigm for differentiating novel AEDs in a model of pharmacoresistant bursting.

Neurotensin receptors have been studied as molecular targets for the treatment of pain, schizophrenia, addiction, or cancer. Neurotensin (NT) and Contulakin-G, a glycopeptide isolated from a predatory cone snail Conus geographus, share a sequence similarity at the C-terminus, which is critical for activation of neurotensin receptors. Both peptides are potent analgesics, although affinity and agonist potency of Contulakin-G toward neurotensin receptors are significantly lower, as compared to those for NT. In this work, we show that the weaker agonist properties of Contulakin-G result in inducing significantly less desensitization of neurotensin receptors and preserving their cell-surface density. Structure-activity relationship (SAR) studies suggested that both glycosylation and charged amino acid residues in Contulakin-G or NT played important roles in desensitizing neurotensin receptors. Computational modeling studies of human neurotensin receptor NTS1 and Contulakin-G confirmed the role of glycosylation in weakening interactions with the receptors. Based on available SAR data, we designed, synthesized, and characterized an analog of Contulakin-G in which the glycosylated amino acid residue, Gal-GalNAc-Thr10, was replaced by memantine-Glu10 residue. This analog exhibited comparable agonist potency and weaker desensitization properties as compared to that of Contulakin-G, while producing analgesia in the animal model of acute pain following systemic administration. We discuss our study in the context of feasibility and safety of developing NT therapeutic agents with improved penetration across the blood-brain barrier. Our work supports engineering peptide-based agonists with diverse abilities to desensitize G-protein coupled receptors and further emphasizes opportunities for conotoxins as novel pharmacological tools and drug candidates.

There are ongoing efforts to develop pain therapeutics with novel mechanisms of action that avoid common side effects associated with other analgesics. The anticonvulsant neuropeptide galanin is a potent regulator of neuronal excitability and has a well established role in pain modulation, making it a potential target for novel therapies. Our previous efforts focused on improving blood-brain-barrier penetration and enhancing the metabolic stability of galanin analogs to protect against seizures. More recently, we designed peripherally acting galanin analogs that reduce pain-related behaviors by acting in the periphery and exhibit preferential binding toward galanin receptor (GalR)2 over GalR1. In this study, we report preclinical studies of a monodisperse oligoethylene glycol–containing galanin analog, NAX 409-9 (previously reported as GalR2-dPEG24), in rodent analgesic and safety models. Results obtained with NAX 409-9 in these tests were compared with the representative analgesics gabapentin, ibuprofen, acetylsalicylic acid, acetaminophen, and morphine. In mice that received intraplantar carrageenan, NAX 409-9 increased paw withdrawal latency with an ED50 of 6.6 mg/kg i.p. NAX 409-9 also increased the paw withdrawal threshold to mechanical stimulation following partial sciatic nerve ligation in rats (2 mg/kg). Conversely, NAX 409-9 had no effect in the tail flick or hot plate assays (up to 24 mg/kg). Importantly, NAX 409-9 did not negatively affect gastrointestinal motility (4–20 mg/kg), respiratory rate (40–80 mg/kg), or bleed time (20 mg/kg). These studies illustrate that this nonbrain-penetrating galanin analog reduces pain behaviors in several models and does not produce some of the dose-limiting toxicities associated with other analgesics.

Although propofol is most commonly known for its general anesthetic properties, at subanesthetic doses, propofol has been effectively used to suppress seizures during refractory status epilepticus, a mechanism, in part, attributed to the inhibition of neuronal sodium channels. In this study, we have designed and synthesized two novel analogs of propofol, HS245 [2-(3-ethyl-4-hydroxy-5-isopropyl-phenyl)-3,3,3-trifluoro-2-hydroxy-propionamide] and HS357 [2-hydroxy-8-(4-hydroxy-3,5-diisopropyl-phenyl)-2-trifluoromethyl-octanoic acid amide], and determined their effects on sodium currents recorded from cultured hippocampal neurons. HS357 had greater affinity for the inactivated state of the sodium channel than propofol and HS245 (0.22 versus 0.74 and 1.2 microM, respectively) and exhibited the greatest ratio of affinity for the resting over the inactivated state. HS357 also demonstrated greater use-dependent block and delayed recovery from inactivation in comparison with propofol and HS245. Under current-clamp conditions, action potentials from hippocampal CA1 neurons in slices were evoked by current injection, or following perfusion with a zero Mg(2+)/7 mM K(+) artificial cerebrospinal fluid solution. Propofol and HS357 reduced the number of current-induced action potentials; however, HS357 caused a greater reduction in the number of spontaneous action potentials. Consistent with these electrophysiology studies, propofol and HS357 protected mice against acute seizures in the 6-Hz (22-mA) partial psychomotor model. Efficacious doses of propofol were associated with an impairment of motor coordination as assessed in the rotorod toxicity assay. In contrast, HS357 demonstrated a 2-fold greater protective index than propofol. Thus, propofol analogs represent an important structural class from which not only effective, but also safer, anti-convulsants may be developed.

In vivo fluctuations in gonadal hormones alter hippocampal excitability and modulate both physiological and pathological hippocampal processes. To assess hormonal effects on excitability within a functional hippocampal circuit, extracellular CA1 field responses were compared in slices from intact male, intact female, orchidectomized male, and ovariectomized female rats. Secondly, the effects of in vitro applications of 17-beta estradiol, progesterone, or testosterone on baseline excitability of slices from gonadectomized rats were assessed versus pre-hormone baseline measures. Finally, using the in vitro kindling model of slice epileptogenesis, steroid hormone effects on interictal-like activity were also examined. Significant sex differences in excitatory postsynaptic potential amplitude were observed, with slices from males having larger excitatory postsynaptic potential amplitudes than those from females. Gonadectomy significantly decreased excitatory postsynaptic potential amplitude in slices from male rats. Slices from gonadectomized male and female rats also showed a decreased dendritic excitatory postsynaptic potential slope relative to slices from intact male and females rats. In vitro application of testosterone significantly increased excitatory postsynaptic potential amplitudes in slices from both orchidectomized males and ovariectomized females and the population spike amplitude of slices from ovariectomized females. Following in vitro kindling, slices from intact males showed greater spontaneous burst rates than slices from intact females, further suggesting an excitatory effect of testosterone. These results suggest: (1) a sex difference in the level of baseline excitability between slices from intact males and females as measured by excitatory postsynaptic potential amplitudes, (2) testosterone has excitatory effects on baseline physiology and kindled hippocampal responses, and (3) slices from males show a greater level of excitability than those from females in the in vitro kindling model.

Viral infection of the CNS can result in encephalitis and acute seizures, increasing the risk for later-life epilepsy. We have previously characterized a novel animal model of temporal lobe epilepsy that recapitulates key sequela in the development of epilepsy following viral infection. C57BL/6 J mice inoculated with the Daniel's strain of Theiler's Murine Encephalomyelitis Virus (TMEV; 3 × 105 PFU, i.c.) display acute limbic seizures that secondarily generalize. A majority of acutely seized animals develop spontaneous seizures weeks to months later. As part of our investigation, we sought to assess behavioral comorbidity following TMEV inoculation. Anxiety, depression, cognitive impairment, and certain psychoses are diagnosed in persons with epilepsy at rates far more frequent than in the general population. We used a battery of behavioral tests to assess anxiety, depression, cognitive impairment, and general health in acutely seized animals inoculated with TMEV and compared behavioral outcomes against age-matched controls receiving a sham injection. We determined that TMEV-seized animals are less likely to move through the exposed center of an open field and are less likely to enter into the lighted half of a light/dark box; both behaviors may be indicative of anxiety-like behavior. TMEV-seized animals also display early and persistent reductions in novel object exploration during novel object place tasks and do not improve in their ability to find a hidden escape platform in Morris water maze testing, indicative of impairment in episodic and spatial memory, respectively. Cresyl violet staining at 35 and 250 days after injection reveals bilateral reductions in hippocampal area, with extensive sclerosis of CA1 evident bilaterally along the rostral–caudal axis. Early and persistent behavioral changes in the TMEV model provide surrogate markers for assessing disease progression as well as endpoints in screening for the efficacy of novel compounds to manage both seizure burden and comorbid conditions.

Delivery of neuropeptides into the central and/or peripheral nervous systems supports development of novel neurotherapeutics for the treatment of pain, epilepsy and other neurological diseases. Our previous work showed that the combination of lipidization and cationization applied to anticonvulsant neuropeptides galanin (GAL) and neuropeptide Y (NPY) improved their penetration across the blood-brain barrier yielding potent antiepileptic lead compounds, such as Gal-B2 (NAX 5055) or NPY-B2. To dissect peripheral and central actions of anticonvulsant neuropeptides, we rationally designed, synthesized and characterized GAL and NPY analogues containing monodisperse (discrete) oligoethyleneglycol-lysine (dPEG-Lys). The dPEGylated analogues Gal-B2-dPEG(24), Gal-R2-dPEG(24) and NPY-dPEG(24) displayed analgesic activities following systemic administration, while avoiding penetration into the brain. Gal-B2-dPEG(24) was synthesized by a stepwise deprotection of orthogonal 4-methoxytrityl and allyloxycarbonyl groups, and subsequent on-resin conjugations of dPEG(24) and palmitic acids, respectively. All the dPEGylated analogues exhibited substantially decreased hydrophobicity (expressed as logD values), increased in vitro serum stabilities and pronounced analgesia in the formalin and carrageenan inflammatory pain assays following systemic administration, while lacking apparent antiseizure activities. These results suggest that discrete PEGylation of neuropeptides offers an attractive strategy for developing neurotherapeutics with restricted penetration into the central nervous system.

Prosthesis of non-critical parts of a polypeptide backbone is an at- tractive strategy to simplify bioactive peptides. This approach was applied to an opi- oid neuropeptide, Met-enkephalin, in which two adjacent Gly2-Gly3 residues were replaced with a series of non-peptidic backbone spacers varying in length and/or physicochemical properties. The backbone spacers did not affect the overall struc- tural properties of the analogues, but they did dramatically reduce their affinities and agonist activities toward - and -opioid receptors. Molecular modeling sug- gested that the decrease of the affinity of Met-enkephalin to -opioid receptor could be accounted for by the loss of a single hydrogen bond. Remarkably, the ana- logues containing the most isostere spacers retained potent antinociceptive and anticonvulsant properties that were comparable to that of the endogenous pep- tide. This unexpected high in vivo potency could not be accounted for by an increase in metabolic stability. Moreover, the antiepileptic activity could not be reversed by opioid receptor antagonists. In summary, the results obtained with the analogues containing backbone spacers suggest a novel mechanism for seizure control in the brain that involves alternative non-opioid signaling.

Galanin is an endogenous neuropeptide that modulates seizures in the brain. Because this neuropeptide does not penetrate the blood-brain barrier, we designed truncated galanin analogues in which nonessential amino acid residues were replaced by cationic and/or lipoamino acid residues. The analogues prevented seizures in the 6 Hz mouse model of epilepsy following intraperitoneal administration. The most active analogue, Gal-B2 (NAX 5055), contained the -Lys-Lys-Lys(palmitoyl)-Lys-NH(2) motif and exhibited high affinity for galanin receptors (K(i) = 3.5 nM and 51.5 nM for GalR1 and GalR2, respectively), logD = 1.24, minimal helical conformation and improved metabolic stability. Structure-activity-relationship analysis suggested that cationization combined with position-specific lipidization was critical for improving the systemic activity of the analogues. Because the anticonvulsant activity of galanin is mediated by the receptors located in hippocampus and other limbic brain structures, our data suggest that these analogues penetrate into the brain. Gal-B2 may lead to development of first-in-class antiepileptic drugs.

Since 1993, the Anticonvulsant Drug Development Program has contributed to the successful development of nine clinically effective drugs for the symptomatic treatment of epilepsy. These include felbamate (1993), gabapentin (1994), lamotrigine (1994), fosphenytoin (1996), topiramate (1996), tiagabine (1997), levetiracetam (1999), zonisamide (2000), and oxcarbazepine (2000). Despite the apparent success of the current discovery process, a significant need persists for more efficacious and less toxic antiepileptic drugs (AEDs). This is particularly true for patients whose seizures remain refractory to the currently available AEDs. This chapter will review the current process for AED discovery employed by the Anticonvulsant Drug Development Program at the University of Utah and other laboratories working toward the common goal of discovering better therapeutic options for patients living with epilepsy. It will discuss some of the inherent advantages and limitations of the primary animal models employed, while offering insight into potential future directions as we seek to better understand the pathophysiology underlying acquired epilepsy, therapy resistance, and epileptogenesis.

Background: Previous studies have demonstrated that methamphetamine abuse leads to memory deficits and these are associated with relapse. Furthermore, extensive evidence indicates that nicotine prevents and/or improves memory deficits in different models of cognitive dysfunction and these nicotinic effects might be mediated by hippocampal or cortical nicotinic acetylcholine receptors. The present study investigated whether nicotine attenuates methamphetamine-induced novel object recognition deficits in rats and explored potential underlying mechanisms. Methods: Adolescent or adult male Sprague-Dawley rats received either nicotine water (10–75 μg/mL) or tap water for several weeks. Methamphetamine (4×7.5mg/kg/injection) or saline was administered either before or after chronic nicotine exposure. Novel object recognition was evaluated 6 days after methamphetamine or saline. Serotonin transporter function and density and α4β2 nicotinic acetylcholine receptor density were assessed on the following day. Results: Chronic nicotine intake via drinking water beginning during either adolescence or adulthood attenuated the novel object recognition deficits caused by a high-dose methamphetamine administration. Similarly, nicotine attenuated methamphetamine-induced deficits in novel object recognition when administered after methamphetamine treatment. However, nicotine did not attenuate the serotonergic deficits caused by methamphetamine in adults. Conversely, nicotine attenuated methamphetamine-induced deficits in α4β2 nicotinic acetylcholine receptor density in the hippocampal CA1 region. Furthermore, nicotine increased α4β2 nicotinic acetylcholine receptor density in the hippocampal CA3, dentate gyrus and perirhinal cortex in both saline- and methamphetamine-treated rats. Conclusions: Overall, these findings suggest that nicotine-induced increases in α4β2 nicotinic acetylcholine receptors in the hippocampus and perirhinal cortex might be one mechanism by which novel object recognition deficits are attenuated by nicotine in methamphetamine-treated rats.