An official website of the United States government
NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.
Noebels JL, Avoli M, Rogawski MA, et al., editors. Jasper's Basic Mechanisms of the Epilepsies. 5th edition. New York: Oxford University Press; 2024. doi: 10.1093/med/9780197549469.003.0066
Recognizing the pressing need for better treatment options for pharmacoresistant patients with epilepsy, collaborative efforts among the NINDS Epilepsy Therapy Screening Program, the pharmaceutical industry, and academia have focused on the inclusion of therapy-resistant animal models for the screening and characterization of novel compounds that offer more effective seizure control for a difficult-to-treat population. This chapter discusses the role of the 6 Hz psychomotor seizure model, the lamotrigine- and phenytoin-resistant kindled rodent models of focal epilepsy, and the post–status epilepticus models of spontaneous recurring seizures in the antiseizure drug discovery process. While each of these models reproduces some aspects of the pathological, behavioral, and pharmacological characteristics of pharmacoresistant seizures in humans, their clinical validity has yet to be established. However, the approval of cenobamate (CBM) in 2019, an ASD with remarkable clinical efficacy (~20% seizure freedom) and profound preclinical efficacy in the 6 Hz test, opens the door to reevaluate these animal seizure models with renewed hope and optimism. While it would have been premature to speculate on the predictive clinical efficacy of CBM based on only the 6 Hz test, it does provide hope that existing animal models can identify new drugs and potentially change the landscape for pharmacoresistance in the future.
In 1937, Putnam and Merrit identified phenytoin from a screen of several hundred hydantoin analogs using the cat maximal electroshock (MES) seizure model (Putnam and Merritt 1937). Since that time, animal seizure and epilepsy models have evolved to include more etiologically relevant models of generalized and focal epilepsy and models of genetic epilepsy. Collectively, these model systems have played an important role in the early identification, characterization, and ultimate development of several new antiseizure medicines (ASMs). As a result of this effort and the concerted partnership among the NINDS Epilepsy Therapy Screening Program (ETSP, previously the Anticonvulsant Screening Program), the pharmaceutical industry, and academia, there have been over two dozen new ASMs brought to the market for the treatment of epilepsy since 1993. These drugs have benefited millions of patients with epilepsy around the world by providing improved efficacy, safety, and tolerability. Unfortunately, there remains a significant unmet need for those patients with epilepsy who fail to achieve complete, or sometimes even adequate, seizure control yet suffer from often burdensome adverse effects of their ASM. Pharmacoresistance to two or more ASMs given at an adequate dose for an adequate amount of time has been estimated to fall between 25% and 40% and has remained at this level for well over 40 years (Kwan and Brodie 2000; Chen, Brodie et al. 2018). Some have argued that the reason underlying this persistent pharmacoresistance is that all the newly approved drugs were initially identified by the same models that have been the mainstay of ASM discovery for 50+ years, including the MES (Putnam and Merritt 1937), subcutaneous pentylenetetrazol seizure model (THORNE 1945), and the kindled rodent (Goddard, McIntyre et al. 1969), that is, “old models identify old drugs.” Given this suggestion and the pressing need for the patient with pharmacoresistant epilepsy, efforts by the NINDS-sponsored ETSP over the last two decades have focused on the inclusion of therapy-resistant animal models into the screening cascade of the University of Utah screening contract (Wilcox, West et al. 2020) in the hope that their inclusion into the discovery process would lead to the identification of more efficacious ASMs for the therapy-resistant patient population. In this chapter, we will focus our comments on the role of the 6 Hz psychomotor seizure model, the lamotrigine (LTG)- and phenytoin (PHT)-resistant kindled rodent models of focal epilepsy, and the post–status epilepticus (SE) models of spontaneous recurring seizures, for example, the systemic kainate and pilocarpine models, the intrahippocampal kainate mouse model of mesial temporal lobe epilepsy, and the basolateral amygdala stimulation model in the ASM discovery process. There are a number of other seizure and epilepsy models that have been developed over the years, including models of traumatic brain injury leading to posttraumatic epilepsy, post-stroke epilepsy models, infection-induced epilepsy models, zebrafish models, and mouse models of genetic epilepsy that have found a niche in ASM discovery, but their value for large-scale medium-throughput screening approaches as models of pharmacoresistant seizures is not currently known and will not be discussed in the present chapter.
Each of the models discussed below has been carefully characterized using several established and clinically approved ASMs and have shown value in their ability to differentiate investigational ASMs. For the purpose of this chapter, we have focused the discussion on 12 of the currently approved ASMs. They include examples of ASMs that enhance GABA-mediated inhibition (PB: phenobarbital and TGB: tiagabine); modulate voltage-gated Na+ channels (PHT: phenytoin; CBZ: carbamazepine; LTG: lamotrigine; and LCM: lacosamide); reduce glutamate-mediated excitatory neurotransmission by blocking AMPA receptors (PER: perampanel); decrease excitability by activating Kv7.2 (EZG: ezogabine/retigabine); modify presynaptic function by binding to synaptic vesicle protein-2A (LEV: levetiracetam); modulate neurotransmitter release by binding to the ɑ2δ subunit of voltage-gated Ca2+ channels (GBP: gabapentin); and exert their antiseizure effects through multiple modes of action (TPM: topiramate; VPA: valproic acid). Where applicable to the discussion, we have also provided some examples of mechanistically interesting drugs that have been found to possess efficacy in one or more of these models but are not approved for the treatment of epilepsy.
The 6 Hz psychomotor seizure is an acutely evoked seizure in mice (Barton, Klein et al. 2001) or rats (Metcalf, West et al. 2017) that has shown promise as a rapid-throughput screening model of pharmacoresistant seizures. Once evoked, the seizure is characterized by a momentary stun, vibrissae twitching, unilateral or bilateral forelimb clonus, and Straub tail. In 1953, it was originally described as a model of “psychomotor seizures” (Brown, Schiffman et al. 1953) but was abandoned as a screening model shortly after its description because of its relative lack of sensitivity to PHT, one of the predominately used ASMs at that time. It was this observation that led Barton and colleagues to reassess the 6 Hz seizure model in 2000 as a potential model of therapy-resistant seizures. Results from these studies confirmed pharmacoresistance to PHT and extended the observation to include other sodium channel-blocking drugs, including LTG, CBZ, and the broad-spectrum ASM, TPM. As it was described by Barton et al. (2001), there is often a shift in the potency of an ASM when the current intensity is increased above that current required to evoke a characteristic seizure in 97% of the mice stimulated; that is, the CC97. For example, at this threshold stimulus intensity, many of the ASMs show efficacy; however, as the intensity is increased to 1.5× CC97 and 2× CC97, the potency and often the protective index (PI: TD50/ED50)1 decline (Table 66–1). A good example of this is provided by LEV. In their study, Barton et al. demonstrated that LEV was effective and reasonably potent (ED50: 4.6 mg/kg) at the CC97 stimulus intensity, but the potency decreased markedly as the intensity of the stimulation current was increased to 1.5× CC97 (ED50: 19.4 mg/kg) and 2× CC97 (ED50: 1089 mg/kg). In contrast to LEV, the monocarbamate, cenobamate (CBM), which received FDA approval for the treatment of partial (focal) onset seizures in adults 18 years of age and older in November 2019, retained its potency in the 6 Hz seizure test as the current intensity was increased from the CC97 (ED50: 11 mg/kg) to 1.5× CC97 (17.9) and 2× CC97 (16.5 mg/kg) (Guignet, Campbell et al. 2020). As noted in Table 66–1, the profile of many of the approved ASMs in the 6 Hz seizure model tends to possess a LEV-like profile; for example, the PI tends to narrow with the higher stimulus intensity, whereas fewer ASMs display a CBM-like profile where the PI remains relatively consistent as the intensity increases. Obviously, the clinical significance of this type of analysis is not clear, but it does suggest that in cases where the efficacy and potency are retained as the severity of the seizure is increased, for example, at higher stimulus intensities, there is a greater possibility that an investigational ASM will be effective at doses devoid of adverse effects.
Antiseizure Medicines Pharmacology of the Mouse and Rat 6 Hz Seizure Model of Pharmacoresistance.
In 2017, Metcalf and colleagues established a rat equivalent of the 6 Hz test in an effort to expand the testing battery of the Utah ETSP contract site and provide another means to differentiate promising ASMs (Metcalf, West et al. 2017). Phenotypically, the 6 Hz seizure in a rat looks similar to that observed in the mouse; however, some differences in the pharmacology of ASMs were noted (Table 66–1). For example, the K+ channel activator, EZG, was more effective in the rat model than in the mouse, whereas most of the Na+ channel modulating drugs, including CBZ, LCM, and PHT, displayed a larger PI in the mouse than the rat at the higher stimulus intensity of 2× CC97. The GABA uptake inhibitor, TGB, also possessed a more favorable PI in the mouse than the rat at the higher stimulus intensity. What is clear is that the 6 Hz test, whether it is conducted in the mouse or rat, displays a degree of pharmacoresistance to the current standards of care and allows an investigator an opportunity to differentiate their investigational drug on the basis of efficacy and potency (Metcalf, West et al. 2017). Whether efficacy in the 6 Hz test will identify a new generation of ASMs for pharmacoresistant epilepsy has yet to be established because no single drug has been brought to the clinic solely on the basis of efficacy in this model.
Kindling is the process that results when an animal is exposed to repeated subconvulsive stimulations delivered to a limbic brain structure such as the amygdala, hippocampus, or piriform cortex (Goddard, McIntyre et al. 1969). With repeated stimulation, the subconvulsive current produces a focal seizure that eventually generalizes to produce a generalized tonic-clonic seizure. In contrast to the acute 6 Hz seizure model, the kindling model is more labor-intensive and thus more expensive to conduct. However, kindling does represent a chronic model of network hyperexcitability, and in this respect it is more closely aligned to actual epilepsy; albeit, spontaneous recurrent seizures are rare in the kindling model unless the animal is “overkindled” (Michalakis, Holsinger et al. 1998).
In the rat amygdala kindling model, it has been known for some time that exposure to a low dose of LTG during the kindling process results in a state of pharmacoresistance to lamotrigine (LTG) in the fully kindled animal (Postma, Krupp et al. 2000). Similarly, the same phenomenon has been observed in animals kindled using the chemoconvulsant pentylenetetrazol (Singh, Pillai et al. 2014). In an effort to develop the LTG-resistant amygdala-kindled rat as a screening tool, subsequent investigations confirmed cross tolerance to CBZ, but not VPA (Srivastava and White 2013).
In 2019, Metcalf and colleagues further refined the pharmacological profile of the LTG-resistant kindled rat to include the Na+ channel blockers eslicarbazepine, LCM, PHT, and rufinamide (Table 66–2; Metcalf, Huff et al. 2019). In contrast to the Na+ channel modulating drugs, ASMs that target GABA receptors, that is, clobazam, clonazepam PB, and GABA uptake (TGB), were found to produce dose-dependent efficacy in the LTG-resistant kindled rat (Metcalf, Huff et al. 2019). Like the Na+ channel blockers, the T-type Ca2+ channel blocker ethosuximide (ETX), the SV2A ligand LEV, and the broad-spectrum ASM TPM were all without effect in the LTG-resistant amygdala-kindled rat. In contrast, the Ca2+ channel ɑ2δ modulator GBP displayed modest efficacy and the KV7.2/3 activators EZG and VPA were highly effective (see Metcalf, Huff et al. 2019, for full discussion). Of the 12 drugs highlighted in this chapter and listed in Table 66–2, the only ASMs to display efficacy at doses devoid of behavioral impairment, that is, PI > 1, were those that increase GABA-mediated inhibition (PB and TGB), EZG/RTG (Kv7.2/7.3 modulator), and VPA, the broad-spectrum ASM. Based on this comprehensive pharmacological profile, it is clear that the LTG-resistant amygdala-kindled rat displays a profile consistent with multidrug pharmacoresistance that supports its utility as a moderate-throughput screening model to further differentiate investigational ASDs.
Antiseizure Medicines Pharmacology of the Lamotrigine (LTG)-Resistant Amygdala-Kindled Rat Model of Pharmacoresistance.
In an effort to develop a less labor-intensive mouse equivalent to the LTG-resistant amygdala-kindled rat, Koneval et al. (2018) characterized the pharmacological profile of mice kindled by repeated corneal stimulation (60 Hz for 3 sec delivered through silver-coated corneal electrodes) in the presence of a low dose of LTG. In their evaluation, they observed that corneal kindled mice (CKM), kindled in the presence of a low dose of LTG, develop a similar resistance to LTG, CBZ, and PHT. Whereas, in contrast to the LTG-resistant amygdala-kindled rat, the LTG-resistant CKM was resistant to the KV7.2/3 activator, EZG, the benzodiazepine, diazepam, and to a modest extent, the broad-spectrum ASM, VPA (Koneval, Knox et al. 2018). These findings demonstrate that the LTG-resistant CKM, much like the LTG-resistant kindled rat, displays a pharmacological profile consistent with pharmacoresistance.
From a drug screening perspective, the LTG-resistant CKM offers several advantages over the rat model. For example, mice, because of their lower body weight, require less drug substance per dose compared to rats, no surgical implantation of kindling electrodes and post-surgery recovery period, and the kindling procedure itself is less technically demanding. The outcome measures are similar, that is, effect of drug on seizure severity as measured by the Racine Scale (Racine 1972). The one disadvantage of the CKM model is that there is not a way, other than Racine Seizure score, to evaluate the effect of drug on the focal seizure activity. However, in the amygdala-kindled LTG-resistant rat, one can utilize the electrical afterdischarge threshold and duration, in addition to the Racine seizure score, as outcome measures for focal seizure severity.
In 1991, Loscher and Rundfelt described the effect of phenytoin (PHT) and CBZ on the afterdischarge threshold (an increase in threshold is suggestive of a positive effect on focal seizure firing) in two populations of amygdala-kindled rats; that is, those that were consistent responders or consistent nonresponders to a challenge dose of PHT (Löscher and Rundfeldt 1991). Their findings suggested that it was possible to use a population of rats that had been prescreened for their response to PHT as a model of therapy-resistant focal epilepsy. This study was the beginning of a decade of work that would thoroughly characterize the “PHT-resistant amygdala-kindled rat.” Specifically, they demonstrated that the difference in response to PHT was not the result of pharmacokinetics, nor was it the result of electrode placement. Moreover, they found that CBZ was equally effective in all rats regardless of their response to PHT—a finding that demonstrated that it was possible to differentiate the profile of ASMs (Table 66–3).
Antiseizure Medicines Pharmacology of the Phenytoin-Resistant Amygdala Kindling Model of Pharmacoresistance.
In 1999, Ebert et al. suggested that a genetic link could contribute to whether an amygdala-kindled rat would be a “responder” or a “nonresponder” to PHT. In their studies, they used a large cohort of male and female amygdala-kindled Wistar rats and found that some rats were consistent responders (defined by the ability of PHT to consistently elevate the afterdischarge threshold) to a challenge dose of PHT, that is, 75 mg/kg, i.p., whereas some rats never responded to this dose of PHT. Their analysis of data from 158 Wistar rats demonstrated that technical factors, including gender, plasma PHT concentration, kindling parameters, electrode location, or differences in focal histology, season, or ambient atmospheric pressure, did not significantly impact whether a rat responded to PHT, and they concluded that “the observed difference between phenytoin responders and nonresponders may be genetically determined” (see Ebert and Löscher 1999; Ebert, Rundfeldt et al. 1999, for additional details).
Overall, Löscher’s laboratory has found that the population of amygdala-kindled rats segregates into three populations; that is, 20% displayed a consistent antiseizure response to PHT; 20% were consistently nonresponders; and the remaining 60% were considered variable responders (Löscher and Rundfeldt 1991). Based on these findings, Löscher has suggested that the three groups represent/model three separate clinical patient populations; for example, drug-refractory patients (represented by the 20% nonresponder group), the patient population which benefits from improved seizure control but never achieves complete seizure control (i.e., those rats that display variable response), and the patient who benefits from complete seizure control group (i.e., those rats that display consistent antiseizure response).
Subsequent investigations found that TPM (Reissmüller, Ebert et al. 2000), GBP (Löscher, Reissmüller et al. 2000), LEV (Löscher, Reissmüller et al. 2000), LTG (Ebert, Reissmüller et al. 2000), and felbamate (Ebert, Reissmüller et al. 2000) are all effective in raising the afterdischarge threshold of PHT-resistant amygdala-kindled rats; a finding consistent with their clinical efficacy in the patient with pharmacoresistant epilepsy (Table 66–3). It is interesting that LEV was the only ASM found to be more effective in elevating the afterdischarge threshold in the PHT nonresponder population (Table 66–3).
As a model of pharmacoresistance, the PHT-resistant amygdala-kindled rat does indeed offer an opportunity to differentiate the anticonvulsant profile of an ASM and to study the molecular and genetic basis of pharmacoresistance. However, it is extremely labor-intensive and costly to conduct these studies because of the time required to “select” and “confirm” PHT nonresponders. Because of the low yield, that is, only 20% of the kindled population will fall into the nonresponder group, large numbers of rats will have to be kindled to obtain a sufficiently large population required for an in-department pharmacological assessment. These challenges make this model more difficult to use for routine screening of investigational ASMs, but they should not discourage its use in efforts to differentiate a lead drug.
The post–status epilepticus (SE) models of spontaneous recurrent seizures (SRS) exhibit some of the greatest parallels to humans with temporal lobe epilepsy (TLE), including the underlying pathophysiology of the disease and the development of pharmacoresistant seizures. Because of the high level of similarity with the human disease, these models are routinely used in drug discovery to identify novel ASMs, particularly in those animals who are refractory to conventional ASM treatment. In these models, acute administration of either chemoconvulsants or electrical stimulation of specific brain regions via depth electrodes is used to induce seizure activity which quickly becomes self-sustaining and long-lasting, even when the stimulus has been removed. Following cessation of SE, animals typically experience a latent period of days to weeks before developing SRS arising from the temporal lobe (see Gorter, van Vliet et al. 2016, for discussion).
In those models where SE is induced by chemical means, the most commonly used agents include kainic acid (KA), a cyclic analog of L-glutamate and agonist of the ionotropic kainate receptors, and pilocarpine, an agonist of muscarinic acetylcholine receptors (see Lévesque, Avoli et al. 2016, for full discussion). Whether injected systemically as seen with KA and pilocarpine, or locally into the brain like KA, these post-SE models reproduce the electroencephalographic, behavioral, and neuropharmacological features of pharmacoresistant epilepsy. In addition to chemical means, electrical stimulation of the amygdala or hippocampus has also validated post-SE models of chronic epilepsy and has been thoroughly utilized over the years to better understand mechanisms of pharmacoresistance (Brandt, Glien et al. 2003; Brandt, Volk et al. 2004). While each post-SE model has its unique strengths and limitations, all have instrumentally furthered our understanding of the pharmacological profile (Table 66–4) of clinically relevant models of pharmacoresistant epilepsy as we will discuss herein.
Antiseizure Medicines Pharmacology in Post–Status Epilepticus Models.
The intra-amygdala kainate model of TLE was introduced in 1979 (Ben-Ari, Lagowska et al. 1979) and demonstrated that localized injections of kainate into the amygdala of rodents can induce behavioral seizures and neuropathology in the hippocampus similar to those occurring in patients. However, focal electrographic seizures are infrequent in both rats and mice following intra-amygdala KA, making it both time- and labor-intensive for studying the efficacy of different ASMs (Tanaka, Kondo et al. 1988; Welzel, Schidlitzki et al. 2020). Since then, the mesial temporal lobe epilepsy (MTLE) mouse model, that is, induced by intra-hippocampal injections in mice, has emerged as an advantageous model in that mice display highly frequent focal electrographic seizures and therefore only requires short periods of electroencephalogram (EEG) recording to evaluate the efficacy of novel ASMs (Bouilleret, Ridoux et al. 1999; Riban, Bouilleret et al. 2002). Because of its utility, the intrahippocampal mouse has become an integral part of the Differentiation arm of the ETSP screening platform (Wilcox, West et al. 2020), and the pharmacology of this model has been extensively characterized over the years (Riban, Bouilleret et al. 2002; Klein, Bankstahl et al. 2015; Duveau, Pouyatos et al. 2016).
In general, while most ASMs can block generalized convulsive seizures in the MTLE mouse, focal electrographic seizures are much more difficult to treat and display a differential sensitivity to different ASMs with drugs that target GABAergic transmission being more effective than other classes of drugs (Klein, Bankstahl et al. 2015; Duveau, Pouyatos et al. 2016). For example, PB, diazepam (DZP), TGB, and vigabatrin (VGB) are able to suppress spontaneous recurrent hippocampal paroxysmal discharges (HPDs), an objective EEG biomarker of focal seizures, in a dose-dependent manner without signs of behavioral impairment (Duveau, Pouyatos et al. 2016). Conversely, a subset of sodium channel blockers, including CBZ and LTG, only suppress HPDs at doses that cause motor impairment and lethargy in animals, whereas the classical sodium channel blocker, PHT, is unable to block electrographic seizures at clinically relevant doses (Klein, Bankstahl et al. 2015; Duveau, Pouyatos et al. 2016). VPA, PER, EZG, and LEV dose-dependently block HPDs; however, LEV is the only drug that can do this at doses devoid of behavioral impairment (unpublished data; Twele, Bankstahl et al. 2015; Duveau, Pouyatos et al. 2016). In contrast to these ASMs, the investigational ASM padsevonil (3 mg/kg, i.p.), which targets the benzodiazepine-sensitive GABAA receptor and SV2A protein, exerted only a minimal effect in the MTLE model (Leclercq, Matagne et al. 2020)—an effect that is consistent with its less-than-robust effect in a double-blind dose finding Phase II placebo-controlled trial in patients with highly refractory epilepsy (Werhahn, Toledo et al. 2020). The marginal effect in the MTLE model is in contrast to its marked efficacy and potency in other seizure and epilepsy models—that is, the amygdala-kindled rat and mouse 6 Hz test—and suggests that the MTLE model is a more stringent model of pharmacoresistance. Whether efficacy in the MTLE model translates to human efficacy is still unknown; but lack of efficacy of padsevonil is consistent with its negative clinical outcome in a well-controlled clinical study.
In addition to having a high frequency of SRS, the MTLE mouse also demonstrates significant animal-to-animal variation in the response to different ASMs, which is representative of human patients and further supports its use as a relevant model of therapy-resistant epilepsy (Klein, Bankstahl et al. 2015). As noted in the PHT-resistant kindled rat model (see above discussion), animals that respond differently to ASMs allow for the separation of nonresponders versus responders to further evaluate mechanisms of pharmacoresistance and identify novel compounds that are more effective for difficult-to-treat seizures. For instance, previous studies have shown that individual responses vary to PB, DZP, VPA, LEV, and PHT, whereas CBZ was not effective in any of the animals at the doses tested in that study (Klein, Bankstahl et al. 2015). Interestingly, the doses of CBZ in the Klein et al study were significantly lower than what was published by Duveau et al. (40 mg/kg vs. 100 mg/kg), and it would be important for future studies to investigate whether CBZ does show efficacy at these higher doses. Furthermore, those animals that were resistant to one ASM were often found to be resistant to a different ASD, regardless of class or mechanism of action. However, consistent with other studies, most animals did respond to drugs that target GABAergic neurotransmission, including PB and DZP (Klein, Bankstahl et al. 2015).
Taken together, the intra-hippocampal kainate mouse model of MTLE is highly advantageous for screening and identifying more effective ASMs for difficult-to-treat focal seizures. Frequent electrographic seizures allow for shorter EEG recording periods and ultimately shortening the length of time required for screening of antiseizure effects of novel drugs. In addition, this model can differentiate between ASM effects on focal and generalized seizures, and even allows for the selection of pharmacoresistant mice to further screen for more effective ASMs in a population of animals with difficult-to-treat seizures.
When injected systemically, KA or pilocarpine induces SE within approximately 1 hour after administration as characterized by a catatonic posture and automatisms that progress to myoclonic twitching of the head and limbs, followed by severe limbic seizures, rearing, and falling (Sperk, Lassmann et al. 1983; Turski, Cavalheiro et al. 1983). Even though these chemoconvulsants initiate SE via different mechanisms of action, both models reproduce the typical histopathological alterations and the development of spontaneous recurrent seizures seen in the patient (Lévesque, Avoli et al. 2016). Furthermore, the development of SRS in this model is consistent with the clinical presentation (Leidy, Elixhauser et al. 2001; Haut 2006), as evidenced by large animal-to-animal variability in both seizure frequency and severity (see Lévesque, Avoli et al. 2016, for review and discussion). In addition, pharmacological treatment in both the KA and pilocarpine models reproduces many of the features in patients with therapy-resistant epilepsy, including (a) conventional ASMs are only able to suppress seizure activity (i.e., to date, no drug has been shown to have disease-modifying properties in TLE); (b) seizures tend to recur whenever drug treatment has stopped; (c) there will always be a subpopulation of animals that are pharmacoresistant to an ASM even if administered at the maximally tolerated dose (Lévesque, Avoli et al. 2016).
While these parallels to the human condition are a strength of these models, it has also come with its challenges for rapidly screening and identifying more effective ASMs. For instance, the large variability in seizure frequency requires both time- and labor-intensive experiments involving 24/7 video-EEG recording for weeks at a time to accurately determine a drug’s efficacy in attenuating spontaneous seizure activity. These, among other reasons, have contributed to the fact that only a few studies have investigated the pharmacology of ASMs in the systemic KA rat model of chronic epilepsy (Grabenstatter, Ferraro et al. 2005; Grabenstatter, Clark et al. 2007; Grabenstatter and Dudek 2008; Ali, Dua et al. 2012). However, studies from the ETSP (Thomson, Metcalf et al. 2020) have described a practical vehicle/drug crossover screening paradigm that requires only 5 days of treatment monitoring in order to quickly differentiate between the efficacy of prototype ASMs. Furthermore, using this cross-over design with a subsequent drug wash-out period between different treatments allows for the use of the same animals with epilepsy for repeated drug trials (Thomson, Metcalf et al. 2020). Interestingly, unlike the intrahippocampal KA mouse model, predictors of ASM response in the post-KA rat were not dependent on mechanisms of action of the drug. For instance, EZG, a potassium channel activator that has never been tested in a spontaneous seizure model, and PB, a GABAergic ASD, were both highly effective in reducing seizure frequency and resulted in seizure freedom in a majority of animals (≥50%) (Thomson 2020). Other prototype ASMs, including clobazam (CLB), clonazepam (CLZ), CBZ, PER, LEV, GBP, and TPM, all showed moderate efficacy in this model, that is, demonstrated a ~50% a reduction in seizure frequency. While CLB, CLZ, PER, and GBP had never been tested in a chronic epilepsy model prior to this study, the findings with CBZ, LEV, and TPM were all consistent with what has been previously published in either post-KA or pilocarpine models (Leite and Cavalheiro 1995; Glien, Brandt et al. 2002; Grabenstatter, Ferraro et al. 2005). Furthermore, a complete lack of efficacy with LCM, LTG, PHT, VPA, and ETX lends further support that this rodent model demonstrates marked differences in pharmacosensitivity to the clinically available ASMs and provides a promising tool for screening novel compounds for more effective seizure control in a difficult-to-treat population.
The significant animal-to-animal variability in the systemic KA model has traditionally required sufficiently powered studies with large treatment groups to accurately capture the efficacy of different ASMs and has been a hurdle for previous researchers (Grabenstatter, Ferraro et al. 2005; Grabenstatter, Clark et al. 2007; Grabenstatter and Dudek 2008; Ali, Dua et al. 2012). However, using a shortened screening paradigm, and selecting for those animals with more severe epilepsy as was done by Thomson et al., expedites the process of identifying those novel compounds that may be more effective in the difficult-to-treat population (Thomson, Metcalf et al. 2020). Additionally, a major advantage to the ETSP screening paradigm is the ability to quickly identify responders versus nonresponders following treatment with an ASM. While not explicitly examined in the Thomson et al. publication, the ability to perform a rapid, cross-over study design with repeated drug testing in the same animals offers a promising model to further evaluate whether pharmacoresistance to one ASM extends to all ASMs or specific classes of drugs and so on. These will be important questions to answer and greatly add to our knowledge of pharmacoresistance in a chronic epilepsy model.
Another limitation to performing chronic dosing studies in animals with epilepsy is that most ASMs are more rapidly eliminated by rodents than humans (Löscher 2007). Therefore, repeated dosing, often multiple times per day, is required for maintaining effective plasma concentrations over the course of a study and represents a major confound when repeatedly injecting hyperexcitable animals with active epilepsy. Recognizing this limitation in preclinical animal studies, some groups have investigated whether delivery of CBZ in a once-per-day drug-in-food paradigm maintains effective plasma concentrations with chronic dosing in the post-KA rat (Grabenstatter, Clark et al. 2007; Ali, Dua et al. 2012). White and colleagues have expanded on this concept even further to develop an automated medication-in-food delivery system that delivers ASMs on a controlled schedule and in accordance with the pharmacokinetic properties of the drug of interest (Thomson and White 2014). These tools are not only a significant step toward more closely resembling the human condition (e.g., repeated dosing via oral delivery); they have also been used to interrogate how medication adherence impacts seizure control, and ultimately whether improving nonadherence can significantly reduce the pharmacoresistant population (Thomson, Modi et al. 2017, Hill, Thomson et al. 2019, Guignet et al., 2023). To date, these studies have only been performed with CBZ, PER, and LEV; however, further investigation of the pharmacological profile in a nonadherence model would determine whether some drugs are more forgiving than others and establish whether these differences are based on pharmacokinetic or pharmacodynamic properties, further adding to our understanding of ASM efficacy in pharmacoresistant populations.
In addition to in vivo models of pharmacoresistant epilepsy, ex vivo approaches offer an attractive complementary approach to drug screening and have been incorporated into the NINDS ETSP for identifying and differentiating potentially novel ASMs. Brain slices from animals that have experienced KA-induced SE demonstrate seizure-like events (SLEs) and recurrent epileptiform discharges under otherwise normal conditions that have a differential sensitivity to various ASMs (West, Saunders et al. 2018). For instance, sodium channel blockers (e.g., CBZ, PHT, LTG, LCM) and GABAergic drugs (e.g., CLB, PB, TGB, VGB) block SLEs in this model, whereas other drugs such as ETX, LEV, and VPA are completely ineffective at all concentrations tested (West, Saunders et al. 2018). Altogether, this ex vivo screening approach meets the definition of pharmacoresistance and can be a useful screening tool for identifying potentially novel ASMs to further characterize in the in vivo rat model.
Originally described by Turski and Cavalheiro in 1983 (Turski, Cavalheiro et al. 1983), systemic administration of pilocarpine has become one of the most widely used post-SE models of TLE (Curia, Longo et al. 2008). Regardless of its utility, chronic drug delivery studies following pilocarpine have been subject to the same demanding technical and logistical hurdles of requiring long-term video-EEG recordings as the post-KA rat in order to evaluate ASM’s efficacy. Nonetheless, the ASM pharmacology of the pilocarpine rat has been extensively characterized since the mid-1990s with Cavalheiro’s group being one of the first to publish a comprehensive summary. Similar to what has been reported in the post-KA rat, PB confers excellent protection, CBZ only marginal protection, and ETX offers no protection at comparable doses between the different models (Leite and Cavalheiro 1995; Thomson, Metcalf et al. 2020). Additional studies with PB have shown that individual responses vary significantly to PB treatment, with some animals achieving complete seizure freedom, while others demonstrate no response at all (Bankstahl, Bankstahl et al. 2012). Additionally, studies with LEV delivered via osmotic mini pumps to maintain therapeutic drug levels suggest a similar level of protection to that observed in the KA rat (~60% reduction in seizure frequency) (Glien, Brandt et al. 2002). However, a significant individual response to the ASM was identified, even though all animals had comparable plasma concentrations of LEV, suggesting that pharmacoresistance to LEV could not be explained by pharmacokinetics alone (Glien, Brandt et al. 2002). In contrast to the KA rat, PHT significantly reduced seizure burden in pilocarpine animals; however, this was at a dose 5 times greater than what was used following KA and did not come without moderate behavioral impairments (Leite and Cavalheiro 1995). Interestingly, VPA was not found to be protective in the KA rat at 600 mg/kg; however, at a dose of 450 mg/kg, VPA did offer significant protection against post-pilocarpine-induced SRS (Leite and Cavalheiro 1995; Thomson, Metcalf et al. 2020). An important point to consider is that Leite and colleagues only observed animals for seizure activity for <10 hours per week in their study, which is significantly less than the continuous 24/7 video-EEG recordings that are the current standard in the field. These shorter recording windows could potentially miss the large individual differences in seizure frequencies between animals, and not be entirely representative of the antiseizure efficacy of different drugs. Nonetheless, these data suggest that while there are similarities between the two systemic chemoconvulsant models, it is possible that subtle differences may exist and can be used to help differentiate the antiseizure efficacy of novel compounds.
In addition to chemical means, electrical stimulation of the basolateral amygdala (BLA) or afferent pathways of the hippocampus via depth electrodes can be used as post-SE models where >90% of animals will develop SRS (Vicedomini and Nadler 1987; Brandt, Glien et al. 2003; Volk and Löscher 2005). Only the BLA model has been used to study the pharmacology of various ASMs, and few studies have separated individual responses to various ASMs. Similar to the pilocarpine model, there are significant differences in animal responses to drugs such as PB (Brandt, Volk et al. 2004) that cannot be attributed to differences in plasma concentrations of the drug. Furthermore, these models reproduce the necessary qualifications to be deemed a preclinical animal model of pharmacoresistance, that is, a lack of an adequate response to two or more ASMs administered at the maximally tolerated dose (MTD) for an adequate amount of time (Chen, Brodie et al. 2018). For instance, a follow-up investigation in those rats that were resistant to PB treatment suggested that >80% of animals did not respond when subsequently treated with PHT (Bethmann, Brandt et al. 2007). However, subsequent treatment with PHT in those animals that responded to PB also resulted in significant seizure control, for example, >50% reduction in seizure frequency (Bethmann, Brandt et al. 2007). Conversely, separate studies that examined whether PB resistance also extended to LTG found that all PB nonresponders and 60% of PB responders exhibited >75% reduction in seizure frequency when given LTG (Brandt and Löscher 2014), further confirming that the BLA SE model provides great utility in differentiating the efficacy of various ASMs and can be used for identifying novel, more effective therapies.
While each model presents with its own strengths and limitations, in general, animal models of therapy-resistant seizures demonstrate significant utility in differentiating among investigational ASMs in a preclinical setting. A common strength of these models is the ability to reproduce the pathological, behavioral, and pharmacological characteristics of pharmacoresistant seizures in humans. However, the major limitation arises when it comes to predictive clinical validity, or the ability for these models to predict the clinical efficacy of a novel ASM. In other words, no single ASM has made it to the market based on its efficacy in any (or all) of these preclinical therapy-resistant seizure models. Historically, this may in part be due to the difficulties in conducting this type of research, for example, laborious, time-consuming, and expensive experiments that result in a low-yield/low-throughput metrics. However, with the introduction of newer screening models (e.g., LTG-resistant CKM, MTLE mouse), and the modification of older screening platforms (e.g., the ETSP’s condensed screening paradigm in the post-KA rat), it may be worth revisiting the characterization of these prototype ASMs to further validate their clinical utility. These types of studies would provide the opportunity to answer important questions about whether we could have predicted the success of certain drugs based on their efficacy in these preclinical animal models. For instance, could the remarkable clinical efficacy of CBM in patients with highly refractory epilepsy have been predicted by its findings in animal models of therapy-resistant seizures, for example, the simple 6 Hz seizure test?
Positive results from two global, randomized, double-blind, placebo-controlled studies, and a large global, multicenter, open-label safety and pharmacokinetic study with CBM in patients with highly refractory epilepsy (patients were taking 1–3 concomitant ASMs) demonstrated statistically significant reductions in seizure frequency across all seizure types studied, including focal aware motor, focal impaired awareness, and focal to bilateral tonic-clonic seizures. These clinical trials demonstrated improved seizure control in adults with uncontrolled focal-onset seizures taking one to three concomitant ASMs (Krauss, Klein et al. 2020; Sperling, Klein et al. 2020, Loscher and Klein. 2021). In addition to improved seizure control, CBM provided significantly higher seizure freedom rates compared to those reported for patients randomized to receive placebo on top of their concomitant ASMs (Chung, French et al. 2020). In fact, the degree of seizure freedom (e.g., 20%) associated with CBM treatment was well above that that seen for any new ASM brought to the market since 1993 when felbamate (FBM) was approved for the treatment of partial (focal) onset seizures (see Guignet, Campbell et al. 2020; Krauss, Klein et al. 2020; Sperling, Klein et al. 2020, Loscher and Klein, 2021 for review and discussion). As discussed in Guignet et al. (2020), CBM possesses a broad-spectrum antiseizure profile in multiple rodent seizure and epilepsy models, including the MES, scPTZ, sc picrotoxinin, kindled rat and Genetic Absence Epilepsy Rat of Strasbourg (GAERS). What separates CBM’s preclinical profile from that of other broad-spectrum ASMs is its efficacy in the 6 Hz seizure test. As tempting as it is to speculate that CBM’s sustained efficacy and potency in the 6 Hz test regardless of the stimulus intensity would have predicted the marked efficacy seen in clinical trials, it would be premature to do so and additional study of the utility of the model is certainly warranted.
Additionally, while CBMs marked efficacy in the 6 Hz test is remarkable, it has yet to be studied in any other therapy-resistant seizure model discussed herein. Further investigation into a drug like CBM would be imperative for detailing a picture of the clinical validity of these animal models. Importantly, comparing CBM’s preclinical efficacy alongside other ASMs such as LEV, a drug that is very effective in patients with refractory focal epilepsy (Cereghino, Biton et al. 2000; Grant and Shorvon 2000) but does not demonstrate the same level of seizure freedom that is seen with CBM, would prove valuable in a population of animals that are pharmacoresistant to other ASMs. Specifically, a major strength of these models is the ability reproduce the clinical presentation where ~30%–40% of patients are refractory to currently available ASMs, a number that has remained stagnant for the last few decades (Chen, Brodie et al. 2018). By identifying and selecting for animals that do not respond to two ASMs, that is, the textbook definition of pharmacoresistance, it provides the ability to screen for more effective therapies for a difficult-to-treat population. Designing preclinical studies that model human clinical trials in patients with refractory epilepsy would provide valuable insight into the clinical utility of these models and may ultimately change the landscape for predicting the clinical success of new ASMs in reducing the percentage of patients with refractory epilepsy.
While no single animal model can reproduce all the features of the human condition, using a battery of pharmacoresistant animal models captures many of the behavioral, electroencephalographic, and pharmacological phenomena of pharmacoresistant seizures. The findings summarized within support the use of these models to potentially aid in the development of novel ASMs for treating patients that are not controlled by currently available drugs. Finally, TLE and seizure dynamics are complex; that is, there are multiple ways in which a seizure can be initiated in the brain. However, combining the use of multiple preclinical models of pharmacoresistant epilepsy will hopefully identify better therapies.
The example set by CBM’s level of seizure freedom demonstrates that it is possible to find highly effective ASMs using animal seizure and epilepsy models. Whether CBM will change the landscape of pharmacoresistance in the future remains to be determined; only time will tell. What is clear is that CBM appears to offer something different in seizure control beyond what we have seen over 30 years and, in this respect, the “old models” did give the patient with therapy-resistant epilepsy a “novel drug” and renewed hope, and supports the continued approach to ASM discovery.
This is an open access publication, available online and distributed under the terms of a Creative Commons Attribution-Non Commercial-No Derivatives 4.0 International licence (CC BY-NC-ND 4.0), a copy of which is available at https://creativecommons.org/licenses/by-nc-nd/4.0/. Subject to this license, all rights are reserved.
Your browsing activity is empty.
Activity recording is turned off.
See more...
