Biology subjects

Metcalf, C. S.

Publications and source records attributed to Metcalf, C. S..

8 recordsLinked to original sources

bioRxiv2024

Development of a preclinical screening platform for clinically relevant therapy of Dravet syndrome

BackgroundPatients with drug-resistant epilepsy, including Dravet syndrome (DS), are frequently prescribed multiple antiseizure medications (ASMs). Nevertheless, people with DS often have inadequate seizure control, and there is an ongoing unmet clinical need to identify novel therapeutics. As a proof-of-principle study to further validate and characterize the Scn1aA1783V/WT mouse model and identify a drug screening paradigm with face, construct, and predictive value, we assessed the efficacy of subchronic administration of stiripentol add-on to clobazam and valproic acid at clinically relevant doses using the Scn1aA1783V/WT mouse model. MethodsWe evaluated the efficacy of STP add-on to CLB and VPA using hyperthermia-induced and video-EEG monitoring of spontaneous seizure tests following a 14-day treatment. VPA was delivered via osmotic minipump, while STP and CLB were administered via food pellets delivered through automatic feeders. Bioanalytical assays were performed to evaluate drug concentrations in plasma and brain using liquid chromatography-tandem mass spectrometry. ResultsSTP, CLB, N-desmethylclobazam, and VPA all yielded plasma concentrations within the human therapeutic plasma concentrations range. STP added to CLB and VPA significantly elevated the seizing temperatures in the hyperthermia-induced seizure assay. CLB, VPA, and STP coadministration significantly reduced spontaneous seizure frequency compared to CLB and VPA combined. SignificanceThis research lays the groundwork for exploring effective add-on compounds to CLB and VPA in treating DS. The study further highlights the utility of the Scn1aA1783V/WT mice in discovering therapies for DS-associated pharmacoresistant seizures. Key pointsO_LIIntegrating pharmacokinetic studies to guide the selection of doses in preclinical studies to achieve target concentrations comparable to the human therapeutic range is crucial in successfully translating animal drug development studies to clinical use. C_LIO_LISTP add-on to CLB and VPA significantly reduced spontaneous seizure frequency in Scn1aA1783V/WT mice. C_LIO_LIA triple-drug polytherapy approach that mimics the clinical treatment paradigm will be an essential preclinical drug screening strategy for identifying novel investigational compounds for Dravet syndrome. C_LI

pharmacology and toxicology↗

bioRxiv2023

Utilizing an acute hyperthermia-induced seizure test and pharmacokinetic studies to establish optimal dosing regimens in a mouse model of Dravet syndrome

ObjectiveThe current standard of care for Dravet Syndrome includes polytherapy after inadequate seizure control with one or more monotherapy approaches. Treatment guidelines are often based on expert opinions, and finding an optimal balance between seizure control and adverse drug effects can be challenging. This study utilizes efficacy and pharmacokinetic analysis of a second-line treatment regimen that combines clobazam and sodium valproate with an add-on drug as a proof-of-principle approach to establish an effective therapeutic regimen in a preclinical mouse model of Dravet Syndrome. MethodWe evaluated the efficacy of add-on therapies stiripentol, cannabidiol, lorcaserin, or fenfluramine to clobazam and sodium valproate against hyperthermia-induced seizures in Scn1aA1783V/WT mice. Clobazam, N-desmethyl clobazam (an active metabolite of clobazam), sodium valproate, stiripentol, and cannabidiol concentrations were quantified in plasma and brain using liquid chromatography-tandem mass spectrometry for the combinations deemed effective against hyperthermia-induced seizures. The concentration data were used to calculate pharmacokinetic parameters via non-compartmental analysis in Phoenix WinNonLin. ResultsHigher doses of stiripentol or cannabidiol, in combination with clobazam and sodium valproate, were effective against hyperthermia-induced seizures in Scn1aA1783V/WT mice. In Scn1aWT/WT mice, brain clobazam and N-desmethyl clobazam concentrations were higher in the triple-drug combinations than in the clobazam monotherapy. Stiripentol and cannabidiol brain concentrations were greater in the triple-drug therapy than when given alone. InterpretationA polypharmacy strategy may be a practical preclinical approach to identifying efficacious compounds for Dravet Syndrome. The drug-drug interactions between compounds used in this study may explain the potentiated efficacy of some combination therapies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=163 SRC="FIGDIR/small/560653v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@70d9a7org.highwire.dtl.DTLVardef@e18a26org.highwire.dtl.DTLVardef@134c74aorg.highwire.dtl.DTLVardef@8065d_HPS_FORMAT_FIGEXP M_FIG C_FIG Key findingsThe hyperthermia-induced seizure assay in Scn1aA1783V/WT mice can identify efficacious antiseizure medications. A triple-drug administration paradigm was designed that mimics clinical standards for add-on therapies in DS. Stiripentol and cannabidiol were effective against hyperthermia-induced seizures when co-administered with clobazam and sodium valproate. Potential drug-drug interactions between compounds may explain the potentiated antiseizure effect in triple-drug therapies. The triple-drug "add-on" therapy approach may be a valuable preclinical strategy for screening investigational compounds for DS, an assay offered at the ETSP.

pharmacology and toxicology↗

bioRxiv2022

Evaluating the efficacy of prototype antiseizure drugs using a preclinical pharmacokinetic approach

ObjectivePharmacokinetics (PK) of a drug drive its exposure, efficacy, and tolerability. A thorough preclinical PK assessment of antiseizure medications (ASMs) is therefore essential to evaluate the clinical potential. We tested protection against evoked seizures of prototype ASMs in conjunction with analysis of plasma and brain PK as a proof-of-principle study to enhance our understanding of drug efficacy and duration of action using rodent seizure models. MethodsIn vivo seizure protection assays were performed in adult male CF-1 mice and Sprague-Dawley rats. Clobazam (CLB), N-desmethylclobzam (NCLB), carbamazepine (CBZ), carbamazepine-10,11-epoxide (CBZE), valproic acid (VPA), and levetiracetam (LEV) concentrations were quantified in plasma and brain using liquid chromatography-tandem mass spectrometry. Mean concentrations of each analyte were calculated and used to determine PK parameters via non-compartmental analysis in Phoenix WinNonLin. ResultsNCLB concentrations were approximately 10-fold greater than CLB in mice. The antiseizure profile of CLB was partially sustained by NCLB in mice. CLB concentrations were lower in rats than in mice. CBZE plasma exposures were approximately 70% of CBZ in both mice and rats, likely contributing to the antiseizure effect of CBZ. VPA showed a relatively short half-life in both mice and rats, which correlated with a sharp decline in efficacy. LEV had a prolonged brain and plasma half-life, associated with a prolonged duration of action in mice. SignificanceThe study demonstrates the utility of PK analyses for understanding the seizure protection time-course in mice and rats. The data indicate that distinct PK profiles of ASMs between mice and rats likely drive differences in drug efficacy between rodent models. Key PointsO_LIThere exist potential contributions of active metabolites to the efficacy of some ASMs. C_LIO_LIThe utility of preclinical PK assessment of ASM is critical to guide our insight into a drug efficacy profile and provide a framework for subchronic dosing strategies. C_LIO_LISpecies-specific variations in PK profiles of ASMs in rodent models of epilepsy may underpin the differences in antiseizure effect in these models. C_LIO_LIPre-clinical drug screening of ASMs should include a (sub)chronic dosing paradigm to better mimic the dosing regimen in the clinic. C_LI

neuroscience↗

bioRxiv2022

Breathing dysfunction and alveolar damage in a mouse model of Dravet syndrome

ObjectiveThe incidence of Sudden Unexpected Death in Epilepsy (SUDEP) is especially high in those with Dravet syndrome (DS). Risk factors have been identified, but the mechanism(s) by which death occurs is not fully understood. Evidence supports ventilatory dysfunction in the pathophysiology of SUDEP. Understanding specific respiratory patterns present at baseline and after seizures at different ages, as well as the health of lung tissue, will allow us to better understand how sudden death occurs in this population. MethodsWhole body plethysmography (WBP) was used to monitor respiration before and after electrically induced seizure in the Scn1aA1783V/WT mouse model of DS weekly for a period of four weeks. Following the four-week WBP study, lungs from surviving animals were collected and stained with hematoxylin and eosin and Weigerts elastin and the density of tissue and elastin were analyzed. ResultsBreathing was diminished in the DS mouse at baseline and following evoked seizures in younger aged mice (P18-P24), consistent with prolonged post-ictal inspiratory time and low respiratory drive compared to the response seen in older animals. In older DS mice, consisting of those that have survived a critical period for mortality, the response to seizure was more robust and included higher respiratory drive, peak inspiratory and expiratory flow rates, tidal and expiratory volumes, and breathing frequency compared to wild-type and relative to baseline. Alveolar damage was also observed in P46-P52 DS mice. SignificanceDifferences in specific respiratory parameters in younger DS animals, during the time when mortality is greatest, compared to older DS animals (i.e. those that have survived the critical period) may allow us to better understand respiratory differences contributing to SUDEP. Lung tissue damage in DS may also contribute to respiratory dysfunction in SUDEP. KEY POINTSO_LIBaseline respiration is diminished in DS mice compared to wild type. C_LIO_LIElectrically induced seizure produced a different respiratory response in younger DS mouse compared to older DS animals. C_LIO_LIAlveolar septal damage is present in DS mice. C_LIO_LIBaseline and post-ictal breathing dysfunction and inefficient oxygenation and CO2 clearance likely potentiated by lung damage may serve as a potential mechanism by which SUDEP occurs in DS. C_LI

physiology↗

bioRxiv2022

Galanin Analogs Prevent Seizure-Induced Respiratory Arrest

ObjectiveSudden Unexpected Death in Epilepsy (SUDEP) accounts for 20% of mortality in those with recurrent seizures. While risk factors, monitoring systems, and standard practices are in place, the pathophysiology of SUDEP is still not well understood. Better knowledge of SUDEP and its potential mechanisms of action is crucial to reducing risk in this patient population and developing potential treatment options. Clinical studies and animal models of SUDEP suggest that diminished post-ictal respiratory control may be the dominant mechanism contributing to mortality. Recently, it was demonstrated that the depletion of the neuropeptide galanin in the amygdala occurs in human SUDEP. The amygdala plays a key role in the central integration of respiratory signaling; the depletion of galanin may represent a critical change that predisposes individuals to SUDEP. MethodsTo evaluate the potential benefit of enhancing galaninergic signaling as a means to protect against SUDEP, we studied seizure-induced respiratory arrest (S-IRA) following central (intracerebroventricular, intra-amygdala) and systemic (intraperitoneal, subcutaneous) administration of galanin agonists. Seizure naive and seizure experienced (fully kindled) mice were tested. ResultsCentral and systemically-administered galanin analogs protect against S-IRA in naive C57Bl/6J mice. Differential efficacy between receptor subtype-selective analogs varied based on the route of administration. Sub-chronic systemic administration at doses that reduced 6 Hz seizures also protected against S-IRA. Acute treatment benefits also extended to fully kindled mice subjected to tonic extension. SignificanceThese data demonstrate that galanin agonists may be protective against post-ictal respiratory collapse. KEY POINTSO_LICentral and systemic galanin agonists prevent seizure-induced respiratory arrest. C_LIO_LIEfficacy was observed in three separate mouse strains under various experimental conditions. C_LIO_LISub-chronic administration demonstrated galanin agonist protection against respiratory arrest. C_LIO_LIAcute systemic administration also conferred protection against respiratory arrest following tonic extension. C_LIO_LIGalanin analogs may represent a novel potential therapy in SUDEP-susceptible individuals. C_LI

neuroscience↗

bioRxiv2021

Evaluation of spontaneous seizure activity, sex-dependent differences, behavioral comorbidities, and alterations in CA1 neuron firing properties in a mouse model of Dravet Syndrome

Dravet syndrome (DS) is a rare childhood epilepsy disorder resulting in spontaneous, recurrent seizures (SRS) and behavioral co-morbidities. To facilitate the discovery and development of anti-seizure drugs for DS, the contract site of the NINDS Epilepsy Therapy Screening Program (ETSP) has continued to evaluate a mouse model of DS. Scn1aA1783V/WT mice exhibited increased hyperactivity, thigmotaxis, and deficits in nest-building behavior. Ex-vivo brain slice electrophysiology experiments revealed increased excitability of hippocampal CA1 neurons specifically due to increased action potential firing frequency in response to brief depolarizations and decreased frequency of spontaneous GABAergic synaptic events. A video-EEG study revealed mice had on average, 1 seizure per day, with males seizing significantly more frequently than females. Increased proportion of seizure activity occurred during the dark phase of the light/dark cycle in both sexes. While clobazam, a drug commonly prescribed to patients with DS, had no effect on SRS activity at the tested doses, the seizure history and frequency observed in this study aids in determining the sample sizes and experimental timeline needed for adequately powered preclinical drug studies. Overall, this study provides a broad description of the Scn1aA1783V/WT mouse and highlights the utility of this model in therapy discovery.

pharmacology and toxicology↗

bioRxiv2020

Spontaneous recurrent seizures in an intra-amygdala kainate microinjection model of temporal lobe epilepsy are differentially sensitive to antiseizure drugs

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. HighlightsO_LIAn intra-amygdala kainate model of TLE was evaluated for pharmacoresistant seizures C_LIO_LIAdministration of midazolam during status epilepticus did not affect mortality C_LIO_LIModel characteristics were evaluated over a 90-day natural history study C_LIO_LISpontaneous seizures were resistant to phenytoin and carbamazepine C_LIO_LISpontaneous seizures were sensitive to valproic acid, diazepam, and phenobarbital C_LI

neuroscience↗

bioRxiv2020

Development of an Antiseizure Drug Screening Platform for Dravet Syndrome at the NINDS contract site for the Epilepsy Therapy Screening Program

ObjectiveDravet 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 thresholds SignificanceOverall, 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 Points O_LIScn1aA1783V/WT mice have a 50% survival rate and all have hyperthermia-induced seizures. C_LIO_LICommon DS treatments such as CLB and combinatorial therapy of CLB, VPA, and STP increase temperature thresholds in Scn1aA1783V/WT mice. C_LIO_LISodium channel blockers, such as CBZ and LTG, decrease temperature thresholds of Scn1aA1783V/WT mice as predicted. C_LIO_LIScn1aA1783V/WT mice are highly pharmacoresitant to common ASDs C_LIO_LIThe Scn1aA1783V/WT may be a useful preclinical drug screening platform for the treatment of DS. C_LI

pharmacology and toxicology↗