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The role of chronobiology in drug-resistance epilepsy: The potential use of a variability and chronotherapy-based individualized platform for improving the response to anti-seizure drugs

Open ArchivePublished:July 01, 2020DOI:https://doi.org/10.1016/j.seizure.2020.06.032

      Highlights

      • Drug-resistant epilepsy (DRE) occurs in a third of patients.
      • Autonomic- and chronotherapy-associated parameters that contribute to the degree of response to therapy.
      • A personalized-based algorithm built on epilepsy-related signatures, autonomic signals, and chronotherapy is presented for overcoming DRE.

      Abstract

      Despite progress in the development of anti-seizure drugs, drug-resistant epilepsy (DRE) occurs in a third of patients. DRE is associated with poor quality of life and increased risk of sudden, unexplained death. The autonomic nervous system and chronobiology play a role in DRE. In the present paper, we provide a narrative review the mechanisms that underlie DRE and characterize some of the autonomic- and chronotherapy-associated parameters that contribute to the degree of response to therapy. Variability describes the functions of many biological systems, which are dynamic and continuously change over time. These systems are required for responses to continuing internal and external triggers, in order to maintain homeostasis and normal function. Both intra- and inter-subject variability in biological systems have been described. We present a platform, which comprises a personalized-based machine learning closed loop algorithm built on epilepsy-related signatures, autonomic signals, and chronotherapy, as a means for overcoming DRE, improving the response, and reducing the toxicity of current therapies.

      Abbreviations:

      ASD (anti-seizure drugs), DRE (drug resistant epilepsy), ANS (autonomic nervous system), ILAE (international league against epilepsy), P-gp (P-glycoprotein), SNP (single nucleotide polymorphism), ABC (ATP binding cassette), VNS (vagus nerve stimulation), DBS (deep brain stimulation), SSR (sympathetic skin response), RRIV (respiratory rate interval variation), RSA (respiration sinus arrhythmia), HR (heart rate), BP (blood pressure), PGES (post-ictal generalized EEG suppression), HWE (hot water epilepsy), CBZ (carbamazepine), CSP (complex partial seizure), MREG (magnetic resonance encephalography), NIRS (near-infrared spectroscopy), SE (spectral entropy), HRV (heart rate variability), HF (high frequency), LF (low frequency), RMSSD (square root of the mean of the sum of the squares of differences between adjacent RR intervals), SDNN (standard deviation of all consecutive R wave intervals), TLE (temporal lobe epilepsy), CSI (cardio sympathetic index), ApEn (approximate entropy), BDNF (brain derived neurotrophic factor), IGF1 (insulin like growth factor 1), SUDEP (sudden unexpected death in epilepsy), CAP (cholinergic anti-inflammatory pathway), RRI (respiratory rate interval), SCN (suprachiasmatic nuclei), IEA (interictal epileptiform activity), JME (juvenile myoclonic epilepsy), mTLE (mesial TLE), DG (dentate gyrus), IGL (intergeniculate leaflet), AE (absence epilepsy), ISO (infra-slow oscillatory), SPK (spontaneous hippocampal EEG spikes), AWE (adults with epilepsy), SD (sleep deprivation), IED (inter-ictal epileptiform discharges), nPSG (night polysomnography), VGSC (voltage gated sodium channels), GEFS+ (generalized epilepsy with febrile seizures plus), PDF (pigment dispersing factor), REM (rapid eye movement), NREM (non REM), DLMO (dim light melatonin onset), MEQ (morningness eveningness questionnaire), mTOR (mammalian target of rapamycin), ECS (electroconvulsive shock), KA (kainic acid), SEs (status epilepticus), SLA (spontaneous locomotor activity), CRH (corticotropin releasing hormone), ACTH (adrenocorticotropin hormone), CORT (corticosterone), PNES (psychogenic non epileptic seizure), VPA (valproic acid), SRS (spontaneous recurrent seizures), MDR1 (multi drug resistance 1), LTG (lamotrigine), LEV (levetiracetam), TPM (topiramate), TDM (therapeutic drug monitoring), TSC (tuberous sclerosis complex)

      Keywords

      1. Introduction

      Epilepsy is the most severe neurological condition that affects individuals of all ages [
      • Sadr S.S.
      • Javanbakht J.
      • Javidan A.N.
      • Ghaffarpour M.
      • Khamse S.
      • Naghshband Z.
      Descriptive epidemiology: prevalence, incidence, sociodemographic factors, socioeconomic domains, and quality of life of epilepsy: an update and systematic review.
      ,
      • Banerjee P.N.
      • Filippi D.
      • Allen Hauser W.
      The descriptive epidemiology of epilepsy-a review.
      ]. An estimated 50 million people have been diagnosed with epilepsy worldwide, and the incidence is 16–51 new cases per 100,000/year. Despite the increased use of anti-seizure drugs (ASD), drug-resistant epilepsy (DRE) remains uncontrolled in a third of patients. DRE often persists even after treatment with two or more drugs [
      • Heinrich A.
      • Zhong X.B.
      • Rasmussen T.P.
      Variability in expression of the human MDR1 drug efflux transporter and genetic variation of the ABCB1 gene: implications for drug-resistant epilepsy.
      ]. DRE is associated with a poor quality of life, and a high risk of sudden, unexplained death [
      • Golyala A.
      • Kwan P.
      Drug development for refractory epilepsy: the past 25 years and beyond.
      ].
      In the present paper, we present a narrative review of some of the possible mechanisms underlying DRE, describe the role of the autonomic nervous system (ANS) in this disease, and summarize the data on the potential role of chronobiology in epilepsy.
      A platform, which comprises a personalized-based machine-learning algorithm comprising epilepsy-related signatures, autonomic signals, and chronotherapy, is described as a potential approach for overcoming DRE and improving the response to therapies.

      1.1 The problem of drug resistant epilepsy: etiology and current methods for overcoming it

      The International League Against Epilepsy (ILAE) characterized DRE based on two levels [
      • Kwan P.
      • Arzimanoglou A.
      • Berg A.T.
      • et al.
      Definition of drug resistant epilepsy: consensus proposal by the ad hoc Task Force of the ILAE Commission on Therapeutic Strategies.
      ]. Level 1 categorizes the outcome of each therapeutic intervention as either "seizure free" or as management failure. This scheme considers that the patient was treated “adequately,” meaning that the ASD was appropriately chosen, well tolerated, and appropriately administered. Level 2 defines DRE as a failure of two treatments, either as monotherapies or as combination regimens [
      • Kwan P.
      • Brodie M.J.
      Early identification of refractory epilepsy.
      ]. Once DRE occurs, the likelihood that further medication trials will lead to freedom from seizure is in the range of 5%–10% [
      • Kalilani L.
      • Sun X.
      • Pelgrims B.
      • Noack-Rink M.
      • Villanueva V.
      The epidemiology of drug-resistant epilepsy: a systematic review and meta-analysis.
      ].
      Several risk factors for developing DRE have been reported. DRE can be age-related [
      • Widdess-Walsh P.
      • Devinsky O.
      Antiepileptic drug resistance and tolerance in epilepsy.
      ,
      • Heinemann U.
      • Kann O.
      • Remy S.
      • Beck H.
      Novel mechanisms underlying drug resistance in temporal lobe epilepsy.
      ]; neuropsychiatric disorder comorbidities, such as mental retardation, and primary psychiatric disorders increase the risk of epilepsy [
      • Hernandez-Ronquillo L.
      • Adams S.
      • Ballendine S.
      • Tellez-Zenteno J.F.
      Epilepsy in an elderly population: classification, etiology and drug resistance.
      ]. Patients with febrile seizures or status epilepticus (SEs), and patients with abnormal electroencephalogram (EEG), or abnormal neuroimaging, are also at increased risk [
      • Hernandez-Ronquillo L.
      • Adams S.
      • Ballendine S.
      • Tellez-Zenteno J.F.
      Epilepsy in an elderly population: classification, etiology and drug resistance.
      ,
      • Orozco-Hernandez J.P.
      • Quintero-Moreno J.F.
      • Marin-Medina D.S.
      • et al.
      Multivariable prediction model of drug resistance in adult patients with generalized epilepsy from Colombia: a case-control study.
      ,
      • Voll A.
      • Hernandez-Ronquillo L.
      • Buckley S.
      • Tellez-Zenteno J.F.
      Predicting drug resistance in adult patients with generalized epilepsy: a case-control study.
      ].
      The mechanism of DRE is multifactorial including environmental, genetic, and drug-related factors [
      • Depondt C.
      The potential of pharmacogenetics in the treatment of epilepsy.
      ]. The target hypothesis suggests a change in cellular targets of ASD, such as compositional alterations in voltage-gated ion channels and neurotransmitter receptors, which reduce the sensitivity to treatment [
      • Tang F.
      • Hartz A.M.S.
      • Bauer B.
      Drug-resistant epilepsy: multiple hypotheses, few answers.
      ]. Blockade of fast-sodium channels in dentate granule cells by carbamazepine (CBZ) was ablated in the hippocampi of patients with temporal lobe epilepsy (TLE) with resistance to CBZ [
      • Remy S.
      • Gabriel S.
      • Urban B.W.
      • et al.
      A novel mechanism underlying drug resistance in chronic epilepsy.
      ]. Lower sensitivity of GABAA receptors to drugs that bind the benzodiazepine site 1 have been described in a model of epilepsy [
      • Remy S.
      • Beck H.
      Molecular and cellular mechanisms of pharmacoresistance in epilepsy.
      ], and similar findings have been described in the brain tissue of patients with refractory TLE [
      • Loup F.
      • Wieser H.G.
      • Yonekawa Y.
      • Aguzzi A.
      • Fritschy J.M.
      Selective alterations in GABAA receptor subtypes in human temporal lobe epilepsy.
      ]. The neural network hypothesis for a mechanism of epileptogenesis suggests that degeneration and remodeling of neural networks caused by seizures inhibits the drugs from accessing their neuronal targets. The molecular basis of this concept was described in patients with TLE, who form new excitatory circuits because of progressive sprouting. Neurogenesis and astrogliosis contribute to the development of abnormal neural pathways leading to drug resistance [
      • Tang F.
      • Hartz A.M.S.
      • Bauer B.
      Drug-resistant epilepsy: multiple hypotheses, few answers.
      ].
      The multidrug transporter hypothesis is based on the premise that there is an overexpression of efflux transporters in the liver, gut, and kidney that reduces plasma levels of ASD, decreasing the amount that reaches the epileptic focus [
      • Lazarowski A.
      • Czornyj L.
      • Lubienieki F.
      • Girardi E.
      • Vazquez S.
      • D’Giano C.
      ABC transporters during epilepsy and mechanisms underlying multidrug resistance in refractory epilepsy.
      ]. The hypothesis is based on the notion that at the epileptic focus there might be an overexpression of multidrug efflux transporters that induce resistance [
      • Sisodiya S.M.
      • Martinian L.
      • Scheffer G.L.
      • et al.
      Vascular colocalization of P-glycoprotein, multidrug-resistance associated protein 1, breast cancer resistance protein and major vault protein in human epileptogenic pathologies.
      ]. Patients with refractory epilepsy demonstrate high levels of P-glycoprotein (P-gp) in endothelial cells, neurons, and astrocytes, which coincides with low plasma levels of ASD [
      • Lazarowski A.
      • Massaro M.
      • Schteinschnaider A.
      • Intruvini S.
      • Sevlever G.
      • Rabinowicz A.
      Neuronal MDR-1 gene expression and persistent low levels of anticonvulsants in a child with refractory epilepsy.
      ,
      • Robey R.W.
      • Lazarowski A.
      • Bates S.E.
      P-glycoprotein--a clinical target in drug-refractory epilepsy?.
      ,
      • Lazarowski A.
      • Sevlever G.
      • Taratuto A.
      • Massaro M.
      • Rabinowicz A.
      Tuberous sclerosis associated with MDR1 gene expression and drug-resistant epilepsy.
      ,
      • Loscher W.
      • Luna-Tortos C.
      • Romermann K.
      • Fedrowitz M.
      Do ATP-binding cassette transporters cause pharmacoresistance in epilepsy? Problems and approaches in determining which antiepileptic drugs are affected.
      ,
      • Dombrowski S.M.
      • Desai S.Y.
      • Marroni M.
      • et al.
      Overexpression of multiple drug resistance genes in endothelial cells from patients with refractory epilepsy.
      ]. Epilepsy patients with partial responses showed lower plasma levels of phenytoin compared with patients with complete responses, regardless of P-gp levels, and this effect was independent of the dose of phenytoin [
      • Iwamoto T.
      • Kagawa Y.
      • Naito Y.
      • Kuzuhara S.
      • Okuda M.
      Clinical evaluation of plasma free phenytoin measurement and factors influencing its protein binding.
      ]. Increased levels of intestinal P-gp were associated with low CBZ levels in plasma, and increased levels of intestinal MRP2 were associated with high CBZ levels in patients with epilepsy [
      • Simon C.
      • Stieger B.
      • Kullak-Ublick G.A.
      • et al.
      Intestinal expression of cytochrome P450 enzymes and ABC transporters and carbamazepine and phenytoin disposition.
      ]. However, several pre-clinical studies did not support this concept, showing a lack of difference in plasma levels of ASD between responders and non-responders [
      • van Vliet E.A.
      • van Schaik R.
      • Edelbroek P.M.
      • et al.
      Region-specific overexpression of P-glycoprotein at the blood-brain barrier affects brain uptake of phenytoin in epileptic rats.
      ,
      • Brandt C.
      • Bethmann K.
      • Gastens A.M.
      • Loscher W.
      The multidrug transporter hypothesis of drug resistance in epilepsy: proof-of-principle in a rat model of temporal lobe epilepsy.
      ].
      The gene variant hypothesis proposes that variation in genes encoding enzymes which process ASD, or ion channels and neurotransmitter receptors, which are directed by ASD, underlie drug efficacy [
      • Depondt C.
      The potential of pharmacogenetics in the treatment of epilepsy.
      ]. Phenytoin is metabolized by CYP2C9 and CYP2C19, and a correlation between reduced activity alleles of CYP2C9 and a lower dose requirement has been described [
      • Schmidt D.
      • Loscher W.
      New developments in antiepileptic drug resistance: an integrative view.
      ,
      • van der Weide J.
      • Steijns L.S.
      • van Weelden M.J.
      • de Haan K.
      The effect of genetic polymorphism of cytochrome P450 CYP2C9 on phenytoin dose requirement.
      ]. A correlation between intronic single nucleotide polymorphisms (SNP) in SCN1A and the maximum dose of CBZ and phenytoin required has also been proposed [
      • Tate S.K.
      • Depondt C.
      • Sisodiya S.M.
      • et al.
      Genetic predictors of the maximum doses patients receive during clinical use of the anti-epileptic drugs carbamazepine and phenytoin.
      ]. This hypothesis is limited by inconsistencies and poor reproducibility in subsequent studies [
      • Angelopoulou C.
      • Veletza S.
      • Heliopoulos I.
      • et al.
      Association of SCN1A gene polymorphism with antiepileptic drug responsiveness in the population of Thrace, Greece.
      ,
      • Manna I.
      • Gambardella A.
      • Bianchi A.
      • et al.
      A functional polymorphism in the SCN1A gene does not influence antiepileptic drug responsiveness in Italian patients with focal epilepsy.
      ].
      The intrinsic severity hypothesis suggests that DRE is related to the severity of the disease [
      • Rogawski M.A.
      • Johnson M.R.
      Intrinsic severity as a determinant of antiepileptic drug refractoriness.
      ]. This is supported by evidence that high frequency of pre-treatment seizures is a predictor of DRE [
      • Wirrell E.C.
      Predicting pharmacoresistance in pediatric epilepsy.
      ]. On the other hand, a randomized controlled study of 1847 epilepsy patients, which compared immediate vs. deferred treatment with ASD, showed that immediate treatment reduces seizure recurrence in years 1–2; however, long term remission was similar in both groups [
      • Marson A.
      • Jacoby A.
      • Johnson A.
      • et al.
      Immediate versus deferred antiepileptic drug treatment for early epilepsy and single seizures: a randomised controlled trial.
      ]. Only a portion of DRE cases have a genetic etiology.
      Treatment options for DRE are ASD, surgery, neuromodulation, a ketogenic diet, and cannabis. ASD is selected based on the basic principles of epilepsy, and on sex, fertility, age, body weight, drug interactions, and comorbidities. Pharmacological synergistic effects generated by the combination of valproic acid (VPA) and lamotrigine, or the addition of subsequent drugs has a small likelihood of inducing remission [
      • Brodie M.J.
      • Yuen A.W.
      Lamotrigine substitution study: evidence for synergism with sodium valproate? 105 Study Group.
      ,
      • Taing K.D.
      • O’Brien T.J.
      • Williams D.A.
      • French C.R.
      Anti-epileptic drug combination efficacy in an in vitro seizure model - phenytoin and valproate, lamotrigine and valproate.
      ,
      • Stephen L.J.
      • Sills G.J.
      • Brodie M.J.
      Lamotrigine and topiramate may be a useful combination.
      ,
      • Choi H.
      • Heiman G.
      • Pandis D.
      • et al.
      Seizure remission and relapse in adults with intractable epilepsy: a cohort study.
      ]. Early surgery in patients with DRE increases the likelihood of post-surgery remission [
      • Sancho J.
      • Ivanez V.
      • Molins A.
      • Lopez Gomez V.
      • Masramon X.
      • Perez M.
      Changes in seizure severity and quality of life in patients with refractory partial epilepsy.
      ]. Patients with lesions on magnetic resonance imaging (MRI) usually respond better to surgery; however, a normal MRI should not preclude surgical evaluation [
      • Jobst B.C.
      • Cascino G.D.
      Resective epilepsy surgery for drug-resistant focal epilepsy: a review.
      ]. Controlled clinical trials in adults with medically intractable focal seizures treated with responsive neurostimulation (RNS) showed that closed-loop responsive neurostimulation to the seizure focus reduces the frequency of disabling seizures. Median seizure reductions of 75 % after 9 years of treatment was demonstrated [
      • Skarpaas T.L.
      • Jarosiewicz B.
      • Morrell M.J.
      Brain-responsive neurostimulation for epilepsy (RNS((R)) System).
      ].
      For patients who are not candidates for surgery, neuromodulation should be considered. Vagus nerve stimulation (VNS) results in a 50 % decrease in seizures in about 30–40 % of patients [
      • Connor Jr., D.E.
      • Nixon M.
      • Nanda A.
      • Guthikonda B.
      Vagal nerve stimulation for the treatment of medically refractory epilepsy: a review of the current literature.
      ]. Deep brain stimulation (DBS) has shown promising results in small studies [
      • Sprengers M.
      • Vonck K.
      • Carrette E.
      • Marson A.G.
      • Boon P.
      Deep brain and cortical stimulation for epilepsy.
      ]. A ketogenic diet (a high-fat, low-carbohydrate diet) results in urinary ketosis that simulates starvation, while maintaining caloric demand, has been associated with a 50 % reduction in seizures in children with DRE [
      • Kossoff E.H.
      • Zupec-Kania B.A.
      • Auvin S.
      • et al.
      Optimal clinical management of children receiving dietary therapies for epilepsy: updated recommendations of the International Ketogenic Diet Study Group.
      ]. Cannabinoids may assist in DRE. In a study of 74 pediatric patients with DRE, the use of cannabis oil showed an 89 % reduction in the rate of seizures [
      • Tzadok M.
      • Uliel-Siboni S.
      • Linder I.
      • et al.
      CBD-enriched medical cannabis for intractable pediatric epilepsy: the current Israeli experience.
      ]. A recent meta-analysis of observational clinical studies on the treatment of refractory epilepsy with cannabidiol (CBD)-based products reviewed 11 studies describing the results in 670 patients. Two thirds of patients reported improvement in the frequency of seizures, and a higher improvement rate was described in patients treated with CBD-rich extracts than in those treated with purified CBD. Thirty-nine percent of responders reported a "50 % reduction or more in the frequency of seizures" although there were no differences between the two compounds [
      • Pamplona F.A.
      • da Silva L.R.
      • Coan A.C.
      Potential clinical benefits of CBD-rich cannabis extracts over purified CBD in treatment-resistant epilepsy: observational data meta-analysis.
      ]. A CBD-based oral solution has been approved for Dravet and Lennox-Gastaut syndrome [
      • Lazaridis D.
      • Eraikhuemen N.
      • Williams K.
      • Lovince J.
      Treatment of seizures associated with lennox-gastaut and dravet syndromes: a focus on cannabidiol oral solution.
      ].
      Taken together, the high variability between studies suggests multiple mechanisms underlying DRE, making it difficult to control. Increasing drug dosages, changing drugs, brain stimulation, or brain surgery are some of the measures taken in these patients, but with only moderate success [
      • Brodie M.J.
      Pharmacological treatment of drug-resistant epilepsy in adults: a practical guide.
      ,
      • Liu J.T.
      • Liu B.
      • Zhang H.
      Surgical versus medical treatment of drug-resistant epilepsy: a systematic review and meta-analysis.
      ]. The benefits of combination therapy with two or more ASD have been difficult to establish. Rational polytherapy, which combines ASD with differing mechanisms of action in the hope of improved efficacy, has not been consistently proven effective [
      • Nair D.R.
      Management of drug-resistant epilepsy.
      ]. Therefore, there is a need to find ways to overcome adaptation to ASD.

      1.2 The role of the central autonomic network in epilepsy and in drug resistance epilepsy

      The central autonomic network (CAN) comprises the amygdala, anterior insula, anterior cingulate cortex, and orbitofrontal cortex. These regions associate with subcortical regions of the CAN, including the hypothalamus, periaqueductal gray, parabrachial areas in the pons, solitary tract nucleus, and ventrolateral medulla [
      • Benarroch E.E.
      The central autonomic network: functional organization, dysfunction, and perspective.
      ,
      • Benarroch E.E.
      The autonomic nervous system: basic anatomy and physiology.
      ]. Inputs into the CAN include viscerosensory feedback transmitted via the solitary tract nucleus, as well as humoral inputs transmitted via circumventricular parts. The CAN incorporates visceral, humoral, and environmental data and coordinates endocrine, autonomic, and behavioral responses to various triggers. CAN activity is state dependent, and it is also affected by the sleep-wake and circadian cycles.
      Epileptic networks are intimately connected with the CAN. Autonomic symptoms and signs are a common occurrence in seizures, although they are often overshadowed by dominant motor phenomena [
      • Baumgartner C.
      • Lurger S.
      • Leutmezer F.
      Autonomic symptoms during epileptic seizures.
      ]. These features are mediated by cortical discharges transmitted into CAN pathways [
      • Baumgartner C.
      • Lurger S.
      • Leutmezer F.
      Autonomic symptoms during epileptic seizures.
      ,
      • Devinsky O.
      • Perrine K.
      • Theodore W.H.
      Interictal autonomic nervous system function in patients with epilepsy.
      ]. They happen during seizures, and can be associated with other symptoms, and rarely occur as the main seizure manifestation [
      • Baumgartner C.
      • Lurger S.
      • Leutmezer F.
      Autonomic symptoms during epileptic seizures.
      ,
      • Panayiotopoulos C.P.
      Epileptic seizures and their classification.
      ].
      Altered autonomic activity is associated with epilepsy [
      • Erickson T.C.
      Cardiac activity during epileptic seizures.
      ,
      • Russell A.E.
      Cessation of the pulse during the onset of epileptic fits: with remarks on the mechanism of fits.
      ,
      • Gastaut H.
      So-called “psychomotor” and “temporal” epilepsy.
      ]. Studies using both spontaneous and prompted seizures, showed that bradycardia, reduction in skin resistance, augmented esophageal activity, and apnea may occur at the start of partial epilepsy [
      • Van Buren J.M.
      Some autonomic concomitants of ictal automatism; a study of temporal lobe attacks.
      ,
      • Van Buren J.M.
      • Bucknam C.A.
      • Pritchard W.L.
      Autonomic representation in the human orbitotemporal cortex.
      ]. Similar manifestations were described in patients who had seizures induced by electroconvulsive therapy (ECT) [
      • Panayiotopoulos C.P.
      Epileptic seizures and their classification.
      ,
      • Mosier J.M.
      • White P.
      • Grant P.
      • Fisher J.E.
      • Taylor R.
      Cerebroautonomic and myographic changes accompanying induced seizures.
      ]. A noteworthy observation is that in some cases these changes continued during the post ictal phase [
      • Panayiotopoulos C.P.
      Epileptic seizures and their classification.
      ,
      • Mosier J.M.
      • White P.
      • Grant P.
      • Fisher J.E.
      • Taylor R.
      Cerebroautonomic and myographic changes accompanying induced seizures.
      ].
      Several studies have examined the effects of epilepsy on the ANS, through quantifying autonomic functions in patients with epilepsy, both in the ictal/peri-ictal and interictal states, with the autonomic cardiovascular regulation being the most studied. Heart rate variability (HRV) is an indirect measure of the ANS influence on the heart and generally considered to reflect sympatho-vagal balance. In general, an increased HRV reflects a shift toward parasympathetic dominance, whereas a decreased HRV indicates a relative increase in sympathetic activity.
      Most HRV studies have used interictal electrocardiography data. Two meta-analyses showed that despite high heterogeneity between studies, possibly due to diverse sample characteristics, adult patients with generalized epilepsy compared to healthy, age-matched controls, manifest disturbed interictal autonomic function. Twenty-four-hour HRV and awake measures, comprising variance, high frequency (HF), low frequency (LF), and LF/HF ratio, are reduced, signifying a shift toward sympathetic dominance [
      • Lotufo P.A.
      • Valiengo L.
      • Bensenor I.M.
      • Brunoni A.R.
      A systematic review and meta-analysis of heart rate variability in epilepsy and antiepileptic drugs.
      ,
      • Myers K.A.
      • Bello-Espinosa L.E.
      • Symonds J.D.
      • et al.
      Heart rate variability in epilepsy: a potential biomarker of sudden unexpected death in epilepsy risk.
      ]. Reduced HRV and sympathetic predominance are more pronounced in adult patients with TLE, and autonomic disorders are more severe in patients with refractory seizures or hippocampal sclerosis [
      • Persson H.
      • Ericson M.
      • Tomson T.
      Heart rate variability in patients with untreated epilepsy.
      ,
      • Ansakorpi H.
      • Korpelainen J.T.
      • Tanskanen P.
      • et al.
      Cardiovascular regulation and hippocampal sclerosis.
      ]. Hyperventilation has been shown to induce sympathetic over-activation in mesial temporal epilepsy [
      • Assenza G.
      • Mecarelli O.
      • Tombini M.
      • et al.
      Hyperventilation induces sympathetic overactivation in mesial temporal epilepsy.
      ]. A few studies have examined HRV in the peri-ictal state, showing inconsistent results, but there also seems to be a general trend toward sympathetic predominance in all kinds of seizures, most prominently TLE and generalized seizures [
      • Brotherstone R.
      • McLellan A.
      Parasympathetic alteration during sub-clinical seizures.
      ,
      • Jeppesen J.
      • Beniczky S.
      • Fuglsang-Frederiksen A.
      • Sidenius P.
      • Jasemian Y.
      Detection of epileptic-seizures by means of power spectrum analysis of heart rate variability: a pilot study.
      ,
      • Surges R.
      • Scott C.A.
      • Walker M.C.
      Enhanced QT shortening and persistent tachycardia after generalized seizures.
      ]. Postictal HRV is lower and continues for as long as 6 h [
      • Toth V.
      • Hejjel L.
      • Fogarasi A.
      • et al.
      Periictal heart rate variability analysis suggests long-term postictal autonomic disturbance in epilepsy.
      ,
      • Wasterlain C.G.
      • Fujikawa D.G.
      • Penix L.
      • Sankar R.
      Pathophysiological mechanisms of brain damage from status epilepticus.
      ]. The fact that interictal hypometabolism can be observed in the area next to an epileptic center on positron emission tomography (PET) may explain these functional alterations [
      • Theodore W.H.
      • Dorwart R.
      • Holmes M.
      • Porter R.J.
      • DiChiro G.
      Neuroimaging in refractory partial seizures: comparison of PET, CT, and MRI.
      ]. Unfavorable HRV changes in subjects with refractory epilepsy within a 6-year follow-up have been described [
      • Suorsa E.
      • Korpelainen J.T.
      • Ansakorpi H.
      • et al.
      Heart rate dynamics in temporal lobe epilepsy-A long-term follow-up study.
      ]. ASD have been suggested to modify ANS functions. Increased HRV has been described in 24 patients with epilepsy, and was attributed to CBZ [
      • Devinsky O.
      • Perrine K.
      • Theodore W.H.
      Interictal autonomic nervous system function in patients with epilepsy.
      ]. Similar findings have been reported by others [
      • Tomson T.
      • Ericson M.
      • Ihrman C.
      • Lindblad L.E.
      Heart rate variability in patients with epilepsy.
      ].
      The ANS in epilepsy is controlled by brain-derived neurotrophic factor (BDNF) and insulin-like growth factor 1 (IGF-1). Chronic or severe epilepsy and, mainly, TLE may disturb BDNF and IGF-1 signaling in the central ANS, leading to autonomic dysfunction and reduced cerebral autoregulation in subjects with focal epilepsy. During the interictal period, lowered levels of BDNF and IGF-1 are associated with decreased autonomic functions and reduced cerebral autoregulation [
      • Chen S.F.
      • Jou S.B.
      • Chen N.C.
      • et al.
      Serum levels of brain-derived neurotrophic factor and insulin-like growth factor 1 are associated with autonomic dysfunction and impaired cerebral autoregulation in patients with epilepsy.
      ].
      Sudden, unexpected death in epilepsy (SUDEP) is an important reason for mortality associated with epilepsy. The incidence of SUDEP is increased in patients with DRE. Autonomic dysfunction is associated with sudden death irrespective of structural heart disease. Respiratory and cardiovascular dysfunction were proposed as a mechanism for SUDEP [
      • Lee J.
      • Devinsky O.
      The role of autonomic dysfunction in sudden unexplained death in epilepsy patients.
      ]. Studies using HRV measures and MRI have shown that atrophy in the brainstem of patients with epilepsy, impairs autonomic control and may increase the risk of SUDEP [
      • Mueller S.G.
      • Nei M.
      • Bateman L.M.
      • et al.
      Brainstem network disruption: A pathway to sudden unexplained death in epilepsy?.
      ]. Subjects with epilepsy manifest irregularities in both sinoatrial node pacemaker and ventricular repolarizing currents, increasing their risk for serious cardiac arrhythmias. Cardiac arrhythmias in patients with DRE can explain SUDEP in these patients [
      • Ravindran K.
      • Powell K.L.
      • Todaro M.
      • O’Brien T.J.
      The pathophysiology of cardiac dysfunction in epilepsy.
      ]. Most cardiac autonomic and ventricular function irregularities are noted during ictal and postictal periods; interictal epileptogenic activity also promotes autonomic imbalance. Patients with untreated recently identified epilepsy showed alterations in HRV that reflect the sympatho-vagal imbalance during the interictal period, including reduced time domain measures, mean HF values, and an increased mean LF and mean LF/HF parameters, along with subclinical deterioration of left ventricular functions, which are associated with increased cardiac mortality [
      • Tosun O.
      • Karatoprak E.
      Analysis of tissue Doppler parameters and 24-hour heart rate variations in children with newly diagnosed untreated idiopathic epilepsy in interictal period.
      ]. In one study, the risk of myocardial infarction and stroke was higher in patients with epilepsy [
      • Labuz-Roszak B.
      • Pierzchala K.
      Assessment of autonomic nervous system in patients with epilepsy in the interictal state. A pilot study.
      ]. In a single trial, patients with epilepsy showed increased left ventricle stiffness and left ventricle filling pressure, and increased left atrial volume and parameters of ANS dysfunction, including reduced chronotropic index and predicted peak heart rate levels during effort. Autonomic dysfunction can account for 52 % of the stiffness observed [
      • Fialho G.L.
      • Wolf P.
      • Walz R.
      • Lin K.
      Increased cardiac stiffness is associated with autonomic dysfunction in patients with temporal lobe epilepsy.
      ].
      The vagus nerve (VN), a main component of the ANS, also regulates the neuro-endocrine-immune axis via stimulation of the hypothalamic-pituitary adrenal axis and central ANS, and the efferent cholinergic anti-inflammatory pathway (CAP). The CAP exerts an anti-TNF effect via the discharge of acetylcholine at the distal VN and connection of the VN with the spleen through the splenic sympathetic nerve [
      • Bonaz B.
      • Picq C.
      • Sinniger V.
      • Mayol J.F.
      • Clarencon D.
      Vagus nerve stimulation: from epilepsy to the cholinergic anti-inflammatory pathway.
      ]. VNS involves placing a bipolar electrode around the left VN, and intermittently stimulating it with a small pulse generator [
      • Boon P.
      • Vonck K.
      • de Reuck J.
      • Caemaert J.
      Vagus nerve stimulation for refractory epilepsy.
      ,
      • Vonck K.
      • Thadani V.
      • Gilbert K.
      • et al.
      Vagus nerve stimulation for refractory epilepsy: a transatlantic experience.
      ]. VNS does not alter the parasympathetic cardiovagal tone. No differences have been described in markers of parasympathetic cardiovagal tone, or baroreflex sensitivity, between baseline, a 6-month visit, and a final visit. Systolic and diastolic blood pressure (BP) upon 5-min of head-up tilt was increased following VNS implantation [
      • Garamendi I.
      • Acera M.
      • Agundez M.
      • et al.
      Cardiovascular autonomic and hemodynamic responses to vagus nerve stimulation in drug-resistant epilepsy.
      ]. VNS of vagal afferents at increased frequency (20−30 Hz) is beneficial in DRE. Low-frequency (5 Hz) VNS of vagal efferent activates the CAP, leading to an anti-inflammatory effect in inflammatory diseases [
      • Bonaz B.
      • Picq C.
      • Sinniger V.
      • Mayol J.F.
      • Clarencon D.
      Vagus nerve stimulation: from epilepsy to the cholinergic anti-inflammatory pathway.
      ].
      VNS leads to a 50 % seizure reduction in 20–40 % of patients with refractory epilepsy [
      • Handforth A.
      • DeGiorgio C.M.
      • Schachter S.C.
      • et al.
      Vagus nerve stimulation therapy for partial-onset seizures: a randomized active-control trial.
      ,
      A randomized controlled trial of chronic vagus nerve stimulation for treatment of medically intractable seizures. The Vagus Nerve Stimulation Study Group.
      ]. It has been suggested that augmenting parasympathetic effects improves DRE [
      • Fan J.J.
      • Shan W.
      • Wu J.P.
      • Wang Q.
      Research progress of vagus nerve stimulation in the treatment of epilepsy.
      ]. Impaired cardiovascular autonomic regulation has been attributed to the seizure process itself [
      • Hirfanoglu T.
      • Serdaroglu A.
      • Cetin I.
      • et al.
      Effects of vagus nerve stimulation on heart rate variability in children with epilepsy.
      ]. In a study of 21 epilepsy patients, VNS affected both sympathetic and parasympathetic cardiovascular modulation without a negative effect on autonomic cardiovascular regulation. VNS did not change respiratory rate intervals (RRI) and BP values. The LF and HF power of RRI and the LF power of BP were augmented, and the HF phase between RRI and respiration decreased significantly [
      • Stemper B.
      • Devinsky O.
      • Haendl T.
      • Welsch G.
      • Hilz M.J.
      Effects of vagus nerve stimulation on cardiovascular regulation in patients with epilepsy.
      ]. Patients with DRE who were undergoing VNS implantation showed a reduced frequency domain (VLF, LF, HF, TP), time domain (SDNN, RMSSD, pNN50), and nonlinear (SD1, SD2) HRV parameters. Non-responders manifested reduced cardiac autonomic function. Pre-surgical HRV parameters, representing parasympathetic cardiac regulation or vagal tone, correlated with responsiveness to VNS [
      • Liu H.
      • Yang Z.
      • Huang L.
      • Qu W.
      • Hao H.
      • Li L.
      Heart-rate variability indices as predictors of the response to vagus nerve stimulation in patients with drug-resistant epilepsy.
      ].
      Autonomic biofeedback has been proposed as a potential therapeutic target for DRE. This is based on the association seen between cortical excitability and peripheral sympathetic arousal [
      • Nagai Y.
      • Goldstein L.H.
      • Critchley H.D.
      • Fenwick P.B.
      Influence of sympathetic autonomic arousal on cortical arousal: implications for a therapeutic behavioural intervention in epilepsy.
      ]. The rationale behind biofeedback based on the effects of negative cortical potentials which mirror cortical arousal that translates to abnormal excitability in epilepsy [
      • Nagai Y.
      • Critchley H.D.
      • Rothwell J.C.
      • Duncan J.S.
      • Trimble M.R.
      Changes in cortical potential associated with modulation of peripheral sympathetic activity in patients with epilepsy.
      ]. A relatively small clinical trial involving 40 patients with DRE showed a 43 % reduction in seizures after one month of biofeedback treatment [
      • Nagai Y.
      Autonomic biofeedback therapy in epilepsy.
      ]. In subjects with generalized seizures, augmented parasympathetic activity to values above those prior to the seizure were shown. These were followed by a reduction to normal values subsequent to the seizure [
      • Delamont R.S.
      • Julu P.O.
      • Jamal G.A.
      Changes in a measure of cardiac vagal activity before and after epileptic seizures.
      ]. It has been suggested that pre-ictal increase in cardiac parasympathetic activity can serve as an indicator for these seizures [
      • Nilsen K.B.
      • Haram M.
      • Tangedal S.
      • Sand T.
      • Brodtkorb E.
      Is elevated pre-ictal heart rate associated with secondary generalization in partial epilepsy?.
      ,
      • Sevcencu C.
      • Struijk J.J.
      Autonomic alterations and cardiac changes in epilepsy.
      ].

      1.3 Chronobiology plays a role in epilepsy

      Chronobiology is the study of physiological rhythms, examining the timing of behavior in relation to both daily and seasonal environmental cycles [
      • Emerson K.J.
      • Bradshaw W.E.
      • Holzapfel C.M.
      Concordance of the circadian clock with the environment is necessary to maximize fitness in natural populations.
      ]. Circadian rhythms are endogenous biological cycles of 24 h. Biological ‘clocks’ are located in the suprachiasmatic nucleus (SCN) [
      • Schwartz W.J.
      • Reppert S.M.
      • Eagan S.M.
      • Moore-Ede M.C.
      In vivo metabolic activity of the suprachiasmatic nuclei: a comparative study.
      ], as well as in peripheral tissues [
      • Takeda N.
      • Maemura K.
      The role of clock genes and circadian rhythm in the development of cardiovascular diseases.
      ]. The activity of the SCN peaks during the day and acts as a physiological pacemaker driving the circadian rhythm of the organism [
      • Welsh D.K.
      • Logothetis D.E.
      • Meister M.
      • Reppert S.M.
      Individual neurons dissociated from rat suprachiasmatic nucleus express independently phased circadian firing rhythms.
      ]. The circadian system permits adaptation to the environment. There is some evidence showing that intermittent light is pro-convulsive [
      • Cavalheiro E.A.
      • Leite J.P.
      • Bortolotto Z.A.
      • Turski W.A.
      • Ikonomidou C.
      • Turski L.
      Long-term effects of pilocarpine in rats: structural damage of the brain triggers kindling and spontaneous recurrent seizures.
      ,
      • Quigg M.
      • Straume M.
      • Menaker M.
      • Bertram 3rd, E.H.
      Temporal distribution of partial seizures: comparison of an animal model with human partial epilepsy.
      ,
      • Quigg M.
      • Clayburn H.
      • Straume M.
      • Menaker M.
      • Bertram 3rd, E.H.
      Effects of circadian regulation and rest-activity state on spontaneous seizures in a rat model of limbic epilepsy.
      ].
      Epilepsy is controlled by cyclic rhythms, with seizure rates increasing and dropping intermittently, over periods of weeks, months, or years. Most epilepsies are subject to some diurnal influence [
      • Quigg M.
      Circadian rhythms: interactions with seizures and epilepsy.
      ]. Patients with frontal lobe-type epilepsy have more seizures during sleep than those with TLE [
      • Bazil C.W.
      • Walczak T.S.
      Effects of sleep and sleep stage on epileptic and nonepileptic seizures.
      ]. In a study of 167 patients with epilepsy associated with unilateral mesial temporal sclerosis, 735 seizures were evaluated. Higher occurrences of seizures were noted during the periods 08:01-12:00 and 16:01-20:00, with fewer seizures between 0:01 and 4:00 [
      • Passarelli V.
      • Castro L.H.
      Gender and age influence in daytime and nighttime seizure occurrence in epilepsy associated with mesial temporal sclerosis.
      ]. In a large analysis of patients (SeizureTracker cohort), 891 out of 1118 patients (80 %) showed circadian modulation in their seizure rates. This was also observed in 11 out of 12 patients of the NeuroVista cohort. In the SeizureTracker cohort, 7–21 % of 1118 subjects manifested robust circaseptan (weekly) rhythms, with a 7-day period [
      • Karoly P.J.
      • Goldenholz D.M.
      • Freestone D.R.
      • et al.
      Circadian and circaseptan rhythms in human epilepsy: a retrospective cohort study.
      ]. The association of seizure timing with fluctuating rates of interictal epileptiform discharges, the interictal epileptiform activity (IEA) that is a marker of brain irritability, was observed among seizures. In a further study, IEA oscillated with circadian and subject-specific multi-day periodicities. The multidien periodicities were most frequently 20–30 days, and seizures occurred especially during the rising phase of multidien IEA rhythms [
      • Baud M.O.
      • Kleen J.K.
      • Mirro E.A.
      • et al.
      Multi-day rhythms modulate seizure risk in epilepsy.
      ]. Seizures can follow a 24 -h non-random or non-uniform shape. In a study of 544 seizures in 123 consecutive subjects, the seizure-specific times were spread along 3- or 4 -h time blocks during a 24 -h period. Non-uniform distribution of seizures was noted in temporal lobe epilepsy, showing two peaks found in both 3- and 4 -h periods. However, as peak times vary between studies, rhythmic exogenous triggers or environmental/social “zeitgebers” may modulate the 24 -h rhythmicity of seizures [
      • Nzwalo H.
      • Menezes Cordeiro I.
      • Santos A.C.
      • Peralta R.
      • Paiva T.
      • Bentes C.
      24-hour rhythmicity of seizures in refractory focal epilepsy.
      ]. Generalized seizures occur most frequently out of sleep and in older patients. In a study of 71 patients with a total of 223 seizures, sleep/wake seizure spreading predicted tonic-clonic development better than the time of day, with more seizures occurring during sleep. Tonic-clonic seizures are more frequent between 00:00-03:00 and 06:00-09:00 [
      • Ramgopal S.
      • Vendrame M.
      • Shah A.
      • et al.
      Circadian patterns of generalized tonic-clonic evolutions in pediatric epilepsy patients.
      ]. ASD and the use of other medications also modifies the sleep patterns of people with epilepsy [
      • Shvarts V.
      • Chung S.
      Epilepsy, antiseizure therapy, and sleep cycle parameters.
      ].
      Patients with DRE manifest reduced time domain, frequency domain, and non-linear domains of HRV parameters. Inhibition of cardiac autonomic modulations is more pronounced during the night. Most differences in HRV values are noted in the early morning (usually 05:00 or 06:00), corresponding to cardiac autonomic dysfunction and subsequent SUDEP, which occurs during night or early morning [
      • Yang Z.
      • Liu H.
      • Meng F.
      • et al.
      The analysis of circadian rhythm of heart rate variability in patients with drug-resistant epilepsy.
      ].
      The occurrence of myoclonic jerks in the early morning, a sleep/wake cycle dependency, characterizes juvenile myoclonic epilepsy (JME) [
      • Kasteleijn-Nolst Trenite D.G.
      • de Weerd A.
      • Beniczky S.
      Chronodependency and provocative factors in juvenile myoclonic epilepsy.
      ]. Mesial TLE (mTLE) is characterized by a recurring pattern of spontaneous seizures, suggesting a reliance on the endogenous clock. Seizures can interfere with the biological rhythm output including circadian oscillation of body temperature, EEG pattern, locomotor activity, and the transcriptome. Dysregulation of the circadian clock in the hippocampus combined with multiple uncoupled oscillators is associated with the development of seizures [
      • Leite Goes Gitai D.
      • de Andrade T.G.
      • Dos Santos Y.D.R.
      • Attaluri S.
      • Shetty A.K.
      Chronobiology of limbic seizures: potential mechanisms and prospects of chronotherapy for mesial temporal lobe epilepsy.
      ]. mTLE seizures peak in the late afternoon and early evening. Both excitatory and inhibitory alterations in the circadian functions of the dentate gyrus (DG), which controls the generation of limbic seizures, add to this circadian rhythm. In an animal model of mTLE with chronic epilepsy, DG excitability is higher in the afternoon and early evening, underlying the time of day-dependency of seizures [
      • Matzen J.
      • Buchheim K.
      • Holtkamp M.
      Circadian dentate gyrus excitability in a rat model of temporal lobe epilepsy.
      ].
      The intergeniculate leaflet (IGL) of the thalamus regulates the circadian rhythm, and its network is vastly GABAergic, consisting of neuropeptide Y-synthesizing and enkephalinergic neurons. Putative enkephalinergic IGL neurons generate action potentials only during light-on conditions. Absence epilepsy (AE) is characterized by spike-wave discharges in the EEG, induced by hypersynchronous thalamo-cortical oscillations. In an animal model, reduced GABAergic synaptic transmission in the IGL, with a higher firing rate of infra-slow oscillations (ISO) and an irregular response to alterations in continuous lighting, have been reported [
      • Chrobok L.
      • Palus K.
      • Jeczmien-Lazur J.S.
      • Chrzanowska A.
      • Kepczynski M.
      • Lewandowski M.H.
      Disinhibition of the intergeniculate leaflet network in the WAG/Rij rat model of absence epilepsy.
      ]. Disturbed light detection mechanisms in AE support sleep-promoting system insufficiencies and other arousal disturbances seen in these patients. Malfunctioning of the light recognition system from the retina to subcortical visual assemblies, which is dependent on oscillatory functions, has been suggested in patients with AE. In a rat model of AE, altered rhythmic function of the lateral geniculate neurons, with an increase in both the infra-slow and fast oscillatory frequencies, has been shown. An altered reaction to sustained changes in ambient light supported alterations in the IGL neuronal firing [
      • Chrobok L.
      • Palus-Chramiec K.
      • Jeczmien-Lazur J.S.
      • Lewandowski M.H.
      Altered oscillation frequencies in the lateral geniculate complex in the rat model of absence epilepsy.
      ]. In a rat model of TLE, after injury by SE, a persistent phase shift of approximately 12 h emerges and development of chronic spontaneous seizures proceeds. Locally, an impaired circadian input to the hippocampus induced spontaneous hippocampal EEG spike (SPK) phase shift. Alterations in the powers of circadian input produced a phase shift in hippocampal neural function. A stable circadian input is required for maintaining natural circadian phases in the hippocampus. Injury to circadian centers, such as the medial septum, which interrupt this equilibrium and alter the circadian regulation, can induce daily rhythms of seizures [
      • Stanley D.A.
      • Talathi S.S.
      • Parekh M.B.
      • et al.
      Phase shift in the 24-hour rhythm of hippocampal EEG spiking activity in a rat model of temporal lobe epilepsy.
      ].
      Sleep-wake cycle-state dependent alterations in cortical excitability play a role in interictal epileptiform discharges [
      • Steriade M.
      • Contreras D.
      Relations between cortical and thalamic cellular events during transition from sleep patterns to paroxysmal activity.
      ,
      • Malow B.A.
      • Lin X.
      • Kushwaha R.
      • Aldrich M.S.
      Interictal spiking increases with sleep depth in temporal lobe epilepsy.
      ]. Non-REM sleep potentiates seizure occurrence, while REM sleep inhibits it. Sleep disruptions, including poor nighttime sleep and daytime somnolence, are common in subjects with epilepsy [
      • Reddy D.S.
      • Chuang S.H.
      • Hunn D.
      • Crepeau A.Z.
      • Maganti R.
      Neuroendocrine aspects of improving sleep in epilepsy.
      ]. Sleep is controlled by the circadian oscillator, a clock-like mechanism, and the occurrence of epilepsy at diverse Zeitgeber times (ZTs) alters sleep courses differently [
      • Yi P.L.
      • Chen Y.J.
      • Lin C.T.
      • Chang F.C.
      Occurrence of epilepsy at different zeitgeber times alters sleep homeostasis differently in rats.
      ]. Sleep patterns in adults with epilepsy (AWE) are irregular if seizures are uncontrolled or happen during sleep. Obstructive sleep apnea is more common in AWE who are older, male, obese, or whose seizures are uncontrolled [
      • Grigg-Damberger M.M.
      • Ralls F.
      Sleep disorders in adults with epilepsy: past, present, and future directions.
      ]. Sleep deprivation (SD) increases the incidence of interictal epileptiform discharges (IED) compared to TLE. SD increases the instability of morning recovery sleep compared with night polysomnography (n-PSG). An increased instability of morning recovery sleep augments IED yield in SD-EEG in TLE patients [
      • Giorgi F.S.
      • Maestri M.
      • Guida M.
      • et al.
      Cyclic alternating pattern and interictal epileptiform discharges during morning sleep after sleep deprivation in temporal lobe epilepsy.
      ].
      The sleep architecture of a Drosophila voltage-gated sodium channel (VGSC) gene mutant that harbors a human generalized epilepsy with febrile seizures plus (GEFS+) mutation suggests a relationship between sleep and epilepsy [
      • Petruccelli E.
      • Lansdon P.
      • Kitamoto T.
      Exaggerated nighttime sleep and defective sleep homeostasis in a Drosophila knock-in model of human epilepsy.
      ,
      • Papale L.A.
      • Makinson C.D.
      • Christopher Ehlen J.
      • et al.
      Altered sleep regulation in a mouse model of SCN1A-derived genetic epilepsy with febrile seizures plus (GEFS+).
      ]. GEFS+ mutant sleep phenotypes are unaffected by pharmacologic reduction of GABA transmission by CBZ and are alleviated by decreasing GABA receptor expression in wake-promoting pigment dispersing factor (PDF) neurons. The data suggests that an elevated GABAergic transmission to PDF neurons accounts for increased nighttime sleep in GEFS+ mutants. [
      • Petruccelli E.
      • Lansdon P.
      • Kitamoto T.
      Exaggerated nighttime sleep and defective sleep homeostasis in a Drosophila knock-in model of human epilepsy.
      ]. In a rat model, kindled epilepsy was induced at three different ZTs, and sleep-wake functions were analyzed prior to and following a seizure. Expression of PER1 protein in the hypothalamic SNC and the circadian rhythm of sleep instability were advanced by a few hours upon stimulation at ZT6. Changes in sleep circadian rhythm and PER1 oscillation, prompted by ZT6-kindling, were inhibited by a hypocretin receptor antagonist in the SCN. ZT6-kindling stimuli changed the circadian oscillator. ZT0-kindling decreased rapid eye movement (REM) and non-REM (NREM) sleep, mediated by the corticotrophin-releasing hormone. ZT13-kindling increased interleukin-1 and subsequently augmented NREM sleep without changing the sleep-wake fluctuation [
      • Yi P.L.
      • Chen Y.J.
      • Lin C.T.
      • Chang F.C.
      Occurrence of epilepsy at different zeitgeber times alters sleep homeostasis differently in rats.
      ].
      Differences in chronotypes among patients with different epilepsy syndromes have been described. A late chronotype is a risk for circadian misalignment, which impacts the regulation of seizures. Several studies suggested that primary generalized epilepsy subjects are more likely to have a late chronotype phenotype [
      • Kendis H.
      • Baron K.
      • Schuele S.U.
      • Patel B.
      • Attarian H.
      Chronotypes in patients with epilepsy: does the type of epilepsy make a difference?.
      ,
      • Hofstra W.A.
      • Gordijn M.C.
      • van Hemert-van der Poel J.C.
      • van der Palen J.
      • De Weerd A.W.
      Chronotypes and subjective sleep parameters in epilepsy patients: a large questionnaire study.
      ]. Subjects with generalized epilepsy show a higher likelihood of being evening types compared to patients with focal epilepsy or those without epilepsy [
      • Kendis H.
      • Baron K.
      • Schuele S.U.
      • Patel B.
      • Attarian H.
      Chronotypes in patients with epilepsy: does the type of epilepsy make a difference?.
      ,
      • Hofstra W.A.
      • Gordijn M.C.
      • van Hemert-van der Poel J.C.
      • van der Palen J.
      • De Weerd A.W.
      Chronotypes and subjective sleep parameters in epilepsy patients: a large questionnaire study.
      ]. Patients with epilepsy do not manifest the same age-related increase in morningness, which is noted in healthy controls [
      • Kendis H.
      • Baron K.
      • Schuele S.U.
      • Patel B.
      • Attarian H.
      Chronotypes in patients with epilepsy: does the type of epilepsy make a difference?.
      ]. Patients with epilepsy tend to be morning-oriented and perceive themselves as morning types, even though this may not be reflected in their dim light melatonin onset (DLMO) values [
      • Manni R.
      • Cremascoli R.
      • De Icco R.
      • Terzaghi M.
      Chronotype in patients with epilepsy: a controlled study in 60 subjects with late-onset focal epilepsy.
      ]. Data concerning endogenous melatonin secretion indicate that patients with epilepsy tend to have a late circadian phase. Subjective measures of chronotype do not specify an evening-oriented chronotype in epilepsy. However, data concerning endogenous melatonin secretion suggests that these subjects manifest a late circadian phase [
      • Manni R.
      • De Icco R.
      • Cremascoli R.
      • et al.
      Circadian phase typing in idiopathic generalized epilepsy: dim light melatonin onset and patterns of melatonin secretion-semicurve findings in adult patients.
      ]. Circadian rhythms of patients admitted for long term EEG and video monitoring, used measurement of the dim light melatonin onset (DLMO). The data showed that when correlating seizure timing to the circadian phase as measured by the DLMO, temporal seizures occurs in the 6 h before DLMO and frontal seizures mainly in 6–12 h after the DLMO. Temporal and frontal seizures occur in a non-random fashion synchronized to a hormonal marker of the circadian timing system [
      • Hofstra W.A.
      • Gordijn M.C.
      • van der Palen J.
      • van Regteren R.
      • Grootemarsink B.E.
      • de Weerd A.W.
      Timing of temporal and frontal seizures in relation to the circadian phase: a prospective pilot study.
      ].
      Patients with generalized epilepsy show a late circadian phase [
      • Manni R.
      • De Icco R.
      • Cremascoli R.
      • et al.
      Circadian phase typing in idiopathic generalized epilepsy: dim light melatonin onset and patterns of melatonin secretion-semicurve findings in adult patients.
      ]. The mean Morningness-Eveningness Questionnaire (MEQ) score was reduced in subjects with epilepsy compared to those with focal epilepsy (FE), but was not significantly lower than that in healthy controls. The mean DLMO time in subjects with epilepsy was 49 min later than that in healthy subjects. A lower increase in melatonin within the 30 min after DLMO has been described in epilepsy [
      • Manni R.
      • De Icco R.
      • Cremascoli R.
      • et al.
      Circadian phase typing in idiopathic generalized epilepsy: dim light melatonin onset and patterns of melatonin secretion-semicurve findings in adult patients.
      ].
      Associations between circadian rhythm-related transcription factors regulating clock genes and the mTOR (mammalian target of rapamycin) signaling pathway have been suggested. Rhythmic activity of hyperactivated mTOR signaling molecules leads to a circadian augmentation in neuronal excitability. Oscillations of neuronal excitability in the SCN have been proposed to modulate the periodic excitability in the hippocampus via the subiculum by long-range projections [
      • Cho C.H.
      Molecular mechanism of circadian rhythmicity of seizures in temporal lobe epilepsy.
      ].
      Rhythmic patterns in epileptic activity and seizure occurrence correlate with specific conditions and circadian variation in excitatory and inhibitory equilibrium. The core circadian genes BMAL1 and CLOCK affect excitability and seizure threshold [
      • Khan S.
      • Nobili L.
      • Khatami R.
      • et al.
      Circadian rhythm and epilepsy.
      ]. Period1 (Per1) is a clock-oscillating gene product, which regulates the circadian rhythm in the hypothalamic SCN. Per1 is also expressed in additional brain areas including the cerebral cortex, amygdala, and hippocampus, suggesting it is involved in wider cellular functions besides the control of rhythm. Chemical or electrical seizure-inducing neuromodulation controls Per1 expression. Electric convulsive shock (ECS) and kainic acid (KA) promote the expression of Per1 mRNA in the hippocampus and cortex [
      • Eun B.
      • Kim H.J.
      • Kim S.Y.
      • et al.
      Induction of Per1 expression following an experimentally induced epilepsy in the mouse hippocampus.
      ]. In a pre-clinical study of mTLE, the circadian phase and strength of activity were altered in early post-SE and epileptic stages. The acrophase of the spontaneous locomotor activity (SLA) rhythm was delayed throughout epileptogenesis and had a fragmented 24 -h rhythmicity. A protracted active stage length was also documented during the epileptic phase. The expression of Bmal1, Cry1, Cry2, Per1, Per2, and Per3 was altered. The diurnal rhythm of Cry1 and Cry2 was absent in the early post-SE and recovered during the epileptic phase. Per1 and Per2 rhythmic expressions were disturbed in post-SE groups [
      • Matos H.C.
      • Koike B.D.V.
      • Pereira W.D.S.
      • et al.
      Rhythms of core clock genes and spontaneous locomotor activity in post-status epilepticus model of mesial temporal lobe epilepsy.
      ]. Attenuated oscillation of several core clock genes was correlated with disturbed diurnal and circadian rest-activity, and sleep-wake patterns, in Kcna1-null mice. EEG confirmed changes in sleep patterns, with more time spent awake and less time spent asleep [
      • Wallace E.
      • Wright S.
      • Schoenike B.
      • Roopra A.
      • Rho J.M.
      • Maganti R.K.
      Altered circadian rhythms and oscillation of clock genes and sirtuin 1 in a model of sudden unexpected death in epilepsy.
      ]. In a preclinical study performed on the hippocampus of rats with epilepsy, lowered expression of Clock was shown, and was proposed to be associated with epileptogenicity [
      • Santos E.A.
      • Marques T.E.
      • Matos Hde C.
      • et al.
      Diurnal variation has effect on differential gene expression analysis in the Hippocampus of the pilocarpine-induced model of mesial temporal lobe epilepsy.
      ].
      Alterations in hormones that are under circadian modulation, such as prolactin, noradrenaline, and oxytocin, occur in generalized seizures [
      • Meierkord H.
      • Shorvon S.
      • Lightman S.L.
      Plasma concentrations of prolactin, noradrenaline, vasopressin and oxytocin during and after a prolonged epileptic seizure.
      ,
      • Hofstra W.A.
      • de Weerd A.W.
      The circadian rhythm and its interaction with human epilepsy: a review of literature.
      ]. The hypothalamic-pituitary-adrenal axis comprises three hormones – corticotropin releasing hormone (CRH), corticosterone (CORT), and adrenocorticotropin hormone (ACTH), which control endogenous circadian rhythms. CRH potentiates seizures [
      • Ehlers C.L.
      • Henriksen S.J.
      • Wang M.
      • Rivier J.
      • Vale W.
      • Bloom F.E.
      Corticotropin releasing factor produces increases in brain excitability and convulsive seizures in rats.
      ], and ACTH and CORT are used as ASD in certain epileptic syndromes [
      • Baram T.Z.
      • Mitchell W.G.
      • Tournay A.
      • Snead O.C.
      • Hanson R.A.
      • Horton E.J.
      High-dose corticotropin (ACTH) versus prednisone for infantile spasms: a prospective, randomized, blinded study.
      ]. Memory deficits in subjects with DRE may be affected by exposure to cortisol. In a study of 52 adults with DRE, those with reduced memory scores had increased cortisol and a slower decline in afternoon levels [
      • Cano-Lopez I.
      • Hidalgo V.
      • Hampel K.G.
      • et al.
      Cortisol and trait anxiety as relevant factors involved in memory performance in people with drug-resistant epilepsy.
      ]. A diurnal configuration was described for cortisol and HRV, shown by increased levels of physiological arousal in the mornings and decreased levels at night in subjects with epilepsy and psychogenic non-epileptic seizures (PNES) [
      • Novakova B.
      • Harris P.R.
      • Reuber M.
      Diurnal patterns and relationships between physiological and self-reported stress in patients with epilepsy and psychogenic non-epileptic seizures.
      ]. Vasopressin is an efferent neurohormone of the SCN [
      • Kalsbeek A.
      • Buijs R.M.
      Rhythms of inhibitory and excitatory output from the circadian timing system as revealed by in vivo microdialysis.
      ]. Vasopressin function is phase-conserved among diurnal and nocturnal species. A decreased level of melatonin was described in patients with refractory epilepsy and was proposed to be a consequence of the natural course of epilepsy or a result of ASD [
      • Paprocka J.
      • Dec R.
      • Jamroz E.
      • Marszal E.
      Melatonin and childhood refractory epilepsy--a pilot study.
      ]. Pinealectomy causes convulsions in some species, and melatonin partially protects against this pro-convulsive effect. Melatonin also increases the current required to induce epileptic after-discharges in a rat model of epilepsy [
      • Mevissen M.
      • Ebert U.
      Anticonvulsant effects of melatonin in amygdala-kindled rats.
      ]. A diurnal rhythm in melatonin serum levels was described in a study of children with febrile and epileptic seizures [
      • Ardura J.
      • Andres J.
      • Garmendia J.R.
      • Ardura F.
      Melatonin in epilepsy and febrile seizures.
      ].

      1.4 Chronotherapy in epilepsy

      The potential role of chronobiology in epilepsy was proposed as a basis for the development of chronotherapy-based modalities, which may have some benefit in DRE. Chronopharmacology, chronopharmaceutical delivery methods, differential medication dosing, and utilization of zeitgebers, including chronobiotics or light-therapy and desynchronization, have been suggested [
      • Loddenkemper T.
      • Lockley S.W.
      • Kaleyias J.
      • Kothare S.V.
      Chronobiology of epilepsy: diagnostic and therapeutic implications of chrono-epileptology.
      ]. The three features of chronopharmacology are the chronobiology of disease, the pharmacokinetics of the drug and its relation to circadian rhythms, and the interaction between biological rhythms and pharmaceutical action. Personalization to a patient's chronotype can benefit chronopharmacology and chronotherapy. These can be assessed by questionnaires like MEQ or by direct measurements of DLMO, actigraphic factors, cortisol production, and sleep parameters [
      • Hofstra W.A.
      • de Weerd A.W.
      The circadian rhythm and its interaction with human epilepsy: a review of literature.
      ,
      • Hofstra W.A.
      • de Weerd A.W.
      How to assess circadian rhythm in humans: a review of literature.
      ]. VPA shows 24 -h variation in absorption, distribution, and excretion [
      • Ramgopal S.
      • Thome-Souza S.
      • Loddenkemper T.
      Chronopharmacology of anti-convulsive therapy.
      ,
      • Meinardi H.
      • Van Der Kleijn E.
      • Meijer J.W.
      • Van Rees H.
      Absorption and distribution of antiepileptic drugs.
      ,
      • Yoshiyama Y.
      • Nakano S.
      • Ogawa N.
      Chronopharmacokinetic study of valproic acid in man: comparison of oral and rectal administration.
      ]. Volunteers receiving continuous infusion of midazolam showed daily alterations in drug concentrations, with slightly higher levels at night [
      • Klotz U.
      • Reimann I.W.
      Chronopharmacokinetic study with prolonged infusion of midazolam.
      ]. Healthy volunteers receiving bi-daily doses of diazepam showed increased levels of diazepam following the morning rather than the evening dose [
      • Nakano S.
      • Watanabe H.
      • Nagai K.
      • Ogawa N.
      Circadian stage-dependent changes in diazepam kinetics.
      ].
      In a preclinical study, large circadian variations in VPA pharmacokinetics were described, and were suggested to be associated with the circadian rhythm in toxicity, as the ideal tolerance paralleled the time at which it induced the lowermost values of C(max) and AUC. A single dose of VPA was administered and matched for 3 weeks to a 12 -h light (rest span) and a 12 -h dark cycle (activity span). The AUCn(0-infinity) was higher when VPA was injected at 19 h after light onset (HALO) compared to the administration at 7 HALO. Dosing at 7 HALO induced the maximum Cl(T) value, whereas the Cl(T) was slower when VPA was administered at 19 HALO. C(max) and AUCn(0-infinity) have a significant circadian rhythm [
      • Ben-Cherif W.
      • Dridi I.
      • Aouam K.
      • Ben-Attia M.
      • Reinberg A.
      • Boughattas N.A.
      Circadian variation of Valproic acid pharmacokinetics in mice.
      ]. The effect of injection time on tolerance to VPA was studied in mice synchronized under a light-dark cycle (12:12). The best tolerance to VPA was shown when the drug was injected in the second half of the light-rest span, which corresponds to the second half of the night for humans. Following a lethal dose, the surviving mice showed a circadian variation in body temperature and body weight loss, with the least temperature change and weight loss occurring when the VPA was administered at 9 HALO. Lethal toxicity also varied according to circadian dosing-time [
      • Ben-Cherif W.
      • Dridi I.
      • Aouam K.
      • Ben-Attia M.
      • Reinberg A.
      • Boughattas N.A.
      Chronotolerance study of the antiepileptic drug valproic acid in mice.
      ].
      Awakening epilepsy occurs more frequently in the late-night hours while focal motor seizures are more common in the early part of the night. The effects of chronotherapeutic dose schedule of phenytoin and CBZ were studied in a group of patients with blood serum levels at subtherapeutic levels and patients with toxic serum levels. Of the 103 patients in the subtherapeutic level group, 51 patients (STG I) received a constant medication dose and the other 52 patients (STG II) were given the majority of the dose at 20:00. Of the 63 patients in the toxic level group, 31 patients (TG I) received a lower dosage at regular times, while the other 31 patients (TG II) received most of the dose at 20:00. The study showed that patients in the STG II group had therapeutic levels of the drug and improved seizure control than STG I patients, and TG II patients had improved drug tolerance compared with those in the TG I group. The data show that a dosing schedule can improve the effect in diurnally active patients with epilepsy in whom standard treatment regimens failed [
      • Yegnanarayan R.
      • Mahesh S.D.
      • Sangle S.
      Chronotherapeutic dose schedule of phenytoin and carbamazepine in epileptic patients.
      ].
      ASD chronotherapy, termed differential dosing, is well tolerated, safe, and effective in managing recurring seizure patterns in certain epilepsies in children [
      • Manganaro S.
      • Loddenkemper T.
      • Rotenberg A.
      The need for antiepileptic drug chronotherapy to treat selected childhood epilepsy syndromes and avert the harmful consequences of drug resistance.
      ]. Using a clobazam dosing regimen, which is tailored to the timing of patients’ seizures, improves its efficacy in controlling seizures. An increased evening dose regimen as add-on therapy was evaluated in subjects with night-time/early-morning seizures based on an increased rate of seizures (> 80 %) at nighttime. Subjects with differential dosing tolerated an increased median total clobazam dose. The median fraction of the total clobazam dose delivered in the evening was 66 % and was associated with a median seizure reduction of 75 % compared to only 50 % in controls. Overall the data supported that an increased-evening differential dose of clobazam enhanced seizure control in patients with night-time and early-morning seizures, supporting the need for tailoring treatment to individualized chronobiology [
      • Thome-Souza S.
      • Klehm J.
      • Jackson M.
      • et al.
      Clobazam higher-evening differential dosing as an add-on therapy in refractory epilepsy.
      ].
      Melatonin is associated with the control of circadian rhythm and exerts a neuroprotective and anti-seizure effect. In a model of TLE, melatonin improved the seizure-latent period, reduced the incidence of spontaneous recurrent seizures (SRSs), and decreased the circadian rhythm of seizures. Melatonin decreased neuronal damage in the hippocampus and piriform cortex [
      • Petkova Z.
      • Tchekalarova J.
      • Pechlivanova D.
      • et al.
      Treatment with melatonin after status epilepticus attenuates seizure activity and neuronal damage but does not prevent the disturbance in diurnal rhythms and behavioral alterations in spontaneously hypertensive rats in kainate model of temporal lobe epilepsy.
      ]. Agomelatine is an anti-depressant, which functions as an agonist to melatonin MT1 and MT2 receptors, and as an antagonist to the serotonin 5HT2C receptor. It also reduces the depolarization-evoked release of glutamate, induces a neuroprotective action, and has been proposed as a treatment for epilepsy [
      • Vimala P.V.
      • Bhutada P.S.
      • Patel F.R.
      Therapeutic potential of agomelatine in epilepsy and epileptic complications.
      ].
      Differential dosing based on the 24 -h seizure rhythm can improve treatment effectiveness while reducing adverse reactions [
      • Guilhoto L.M.
      • Loddenkemper T.
      • Vendrame M.
      • Bergin A.
      • Bourgeois B.F.
      • Kothare S.V.
      Higher evening antiepileptic drug dose for nocturnal and early-morning seizures.
      ]. Some ASD have side effects which also have a 24 -h rhythmicity, and, therefore, differential dosing may help avoid or reduce these side effects [
      • Ben-Cherif W.
      • Dridi I.
      • Aouam K.
      • Ben-Attia M.
      • Reinberg A.
      • Boughattas N.A.
      Chronotolerance study of the antiepileptic drug valproic acid in mice.
      ].

      1.5 Variability in biological systems and response to ASD

      Variability characterizes the functions of many biological systems that are dynamic and continuously change over time. These systems are mandatory for the responses to internal and external triggers that maintain homeostasis and normal function [
      • de Lorenzo V.
      • Schmidt M.
      Biological standards for the Knowledge-Based BioEconomy: what is at stake.
      ,
      • Gsponer J.
      • Babu M.M.
      The rules of disorder or why disorder rules.
      ,
      • Buckle A.M.
      • Borg N.A.
      Integrating experiment and theory to understand TCR-pMHC dynamics.
      ,
      • Lodygin D.
      • Flugel A.
      Intravital real-time analysis of T-cell activation in health and disease.
      ,
      • Bandiera L.
      • Furini S.
      • Giordano E.
      Phenotypic variability in synthetic biology applications: dealing with noise in microbial gene expression.
      ]. These dynamic systems contain structures that are self-organized according to morphological, biochemical, and environmental constraints in order to identify the most stable state [
      • Ghanjal A.
      • Motaqi M.
      • Arab Z.
      • Hatef B.
      Force variability in the short- and long-term type 2 diabetes mellitus.
      ]. Functional variability has been shown at the intracellular level, as well as at the whole-organ level [
      • Ilan Y.
      Advanced tailored randomness: a novel approach for improving the efficacy of biological systems.
      ,
      • Ilan Y.
      Beta-glycosphingolipids as mediators of both inflammation and immune tolerance: a manifestation of randomness in biological systems.
      ,
      • Ilan Y.
      Randomness in microtubule dynamics: an error that requires correction or an inherent plasticity required for normal cellular function?.
      ,
      • Ilan Y.
      Generating randomness: making the most out of disordering a false order into a real one.
      ]. Both intra- and inter-subject variability have been described at cellular and at whole-organ levels [
      • Ilan Y.
      Generating randomness: making the most out of disordering a false order into a real one.
      ,
      • Ilan Y.
      Randomness in microtubule dynamics: an error that requires correction or an inherent plasticity required for normal cellular function?.
      ,
      • Singh N.
      • Moneghetti K.J.
      • Christle J.W.
      • Hadley D.
      • Froelicher V.
      • Plews D.
      Heart rate variability: an old metric with new meaning in the era of using mHealth technologies for health and exercise training guidance. Part two: prognosis and training.
      ,
      • Lees T.
      • Shad-Kaneez F.
      • Simpson A.M.
      • Nassif N.T.
      • Lin Y.
      • Lal S.
      Heart rate variability as a biomarker for predicting stroke, post-stroke complications and functionality.
      ,
      • Herssens N.
      • Verbecque E.
      • Hallemans A.
      • Vereeck L.
      • Van Rompaey V.
      • Saeys W.
      Do spatiotemporal parameters and gait variability differ across the lifespan of healthy adults? A systematic review.
      ,
      • Henriques T.
      • Munshi M.N.
      • Segal A.R.
      • Costa M.D.
      • Goldberger A.L.
      "Glucose-at-a-Glance": new method to visualize the dynamics of continuous glucose monitoring data.
      ,
      • Tosato F.
      • Bernardi D.
      • Sanzari M.C.
      • Pantano G.
      • Plebani M.
      Biological variability of lymphocyte subsets of human adults’ blood.
      ]. Loss of normal variability is associated with organ dysfunction and is linked to poor prognosis. Loss of normal HRV is linked to high cardiac morbidity and mortality [
      • Ramirez-Villegas J.F.
      • Lam-Espinosa E.
      • Ramirez-Moreno D.F.
      • Calvo-Echeverry P.C.
      • Agredo-Rodriguez W.
      Heart rate variability dynamics for the prognosis of cardiovascular risk.
      ]. Loss of variability in glucose serum levels has been associated with diabetes development [
      • Henriques T.
      • Munshi M.N.
      • Segal A.R.
      • Costa M.D.
      • Goldberger A.L.
      "Glucose-at-a-Glance": new method to visualize the dynamics of continuous glucose monitoring data.
      ]. Therefore, for many biological systems, rules are difficult to identify, and they seem to be working with continuously changing dynamics manifested by a high degree of variability [
      • de Lorenzo V.
      • Schmidt M.
      Biological standards for the Knowledge-Based BioEconomy: what is at stake.
      ,
      • Gsponer J.
      • Babu M.M.
      The rules of disorder or why disorder rules.
      ,
      • Buckle A.M.
      • Borg N.A.
      Integrating experiment and theory to understand TCR-pMHC dynamics.
      ,
      • Lodygin D.
      • Flugel A.
      Intravital real-time analysis of T-cell activation in health and disease.
      ].
      In epilepsy, changes noted in seizure occurrences do not reflect alterations in the risks for the development of seizures. Alterations in seizure incidence occur due to probabilistic variation in seizure risks initiated by normal historical fluctuations, leading to unpredictability in seizure events. No statistical approach can distinguish predictable changes in the incidence of seizure events due to natural variability from changes in the underlying risks for these events. Using a database of 1.2 million recorded seizure events over 8 years, an epilepsy seizure risk assessment tool (EpiSAT), which uses a Bayesian mixed-effects hidden Markov model, was developed. The score was able to predict alterations in the risk for seizure events using a subject-reported seizure diary and clinical information, and differentiate real changes in the risk from natural variations in the frequency of seizures [
      • Chiang S.
      • Vannucci M.
      • Goldenholz D.M.
      • Moss R.
      • Stern J.M.
      Epilepsy as a dynamic disease: a Bayesian model for differentiating seizure risk from natural variability.
      ]. Chronic EEG studies indicated that epilepsy is a cyclic disease with rhythms operating over many different time scales: multi-day (multidien), circadian, and seasonal. However, seizure events were not evenly spread over time, and were clustered at several periods. The multidien rhythms were not synchronous across animals, suggesting an endogenous generator [
      • Baud M.O.
      • Ghestem A.
      • Benoliel J.J.
      • Becker C.
      • Bernard C.
      Endogenous multidien rhythm of epilepsy in rats.
      ]. Prediction of a seizure using chaos analysis of signals from EEG has been proposed. A chaos attractor was reconstructed in the phase space of EEG. Entropy parameters calculated from the Lyapunov exponents showed that prior to the seizure, these parameters had lower values when compared with healthy conditions [
      • Yambe T.
      • Asano E.
      • Mauyama S.
      • et al.
      Chaos analysis of electro encephalography and control of seizure attack of epilepsy patients.
      ].
      Inter- and intra-individual differences in the response to medications are attributed to pharmacogenomics- and pharmacodynamics-based drug metabolism [
      • Leino A.D.
      • King E.C.
      • Jiang W.
      • et al.
      Assessment of tacrolimus intrapatient variability in stable adherent transplant recipients: establishing baseline values.
      ,
      • Gueta I.
      • Markovits N.
      • Yarden-Bilavsky H.
      • et al.
      High tacrolimus trough level variability is associated with rejections after heart transplant.
      ,
      • Gueta I.
      • Markovits N.
      • Yarden-Bilavsky H.
      • et al.
      Intrapatient variability in tacrolimus trough levels after solid organ transplantation varies at different postoperative time periods.
      ,
      • Del Bello A.
      • Congy-Jolivet N.
      • Danjoux M.
      • et al.
      High tacrolimus intra-patient variability is associated with graft rejection, and de novo donor-specific antibodies occurrence after liver transplantation.
      ]. The high degree of variability in many systems is also reflected in the drug response. Both inter- and intra-patient variabilities, which are beyond the expected pharmacodynamics and pharmacokinetics of the medications, have been described [
      • Murray B.W.
      • Miller N.
      Durability of kinase-directed therapies--a network perspective on response and resistance.
      ,
      • Urquhart B.L.
      • Tirona R.G.
      • Kim R.B.
      Nuclear receptors and the regulation of drug-metabolizing enzymes and drug transporters: implications for interindividual variability in response to drugs.
      ,
      • Theken K.N.
      Variability in analgesic response to non-steroidal anti-inflammatory drugs.
      ,
      • Bruno A.
      • Tacconelli S.
      • Patrignani P.
      Variability in the response to non-steroidal anti-inflammatory drugs: mechanisms and perspectives.
      ,
      • Elgart V.
      • Lin J.R.
      • Loscalzo J.
      Determinants of drug-target interactions at the single cell level.
      ]. This high degree of variability also reflects the unpredictability of the response, which is noted in many patients with chronic diseases [
      • Kenig A.
      • Ilan Y.
      A personalized signature and chronotherapy-based platform for improving the efficacy of Sepsis treatment.
      ,
      • Khoury T.
      • Ilan Y.
      Introducing patterns of variability for overcoming compensatory adaptation of the immune system to immunomodulatory agents: a novel method for improving clinical response to Anti-TNF therapies.
      ].
      The heterogeneity in the response to drugs has been described at the single-cell level [
      • Ilan Y.
      Generating randomness: making the most out of disordering a false order into a real one.
      ,
      • Khoury T.
      • Ilan Y.
      Introducing patterns of variability for overcoming compensatory adaptation of the immune system to immunomodulatory agents: a novel method for improving clinical response to Anti-TNF therapies.
      ,
      • El-Haj M.
      • Kanovitch D.
      • Ilan Y.
      Personalized inherent randomness of the immune system is manifested by an individualized response to immune triggers and immunomodulatory therapies: a novel platform for designing personalized immunotherapies.
      ]. Interactions of the drug with its cellular target cannot always be defined by simple diffusion and intrinsic chemical reactions. In many systems, multiple non-specific interactions between macromolecules in cells and medications are involved in determining the effect. These are not considered by simple pharmacodynamics and pharmacokinetic rules. The lack of specificity has been shown to be associated with a slow incorporation in the kinetics of DNA-binding drugs. Non-specific interactions have been demonstrated in several cellular compartments, as shown by a high degree of variability in intracellular drug kinetics [
      • Elgart V.
      • Lin J.R.
      • Loscalzo J.
      Determinants of drug-target interactions at the single cell level.
      ]. A marked daily variability in drug serum levels has been described for a single subject. This intra-patient variability further supports the notion that this is a complex dynamic system reflected by a high level of unpredictability [
      • Del Bello A.
      • Congy-Jolivet N.
      • Danjoux M.
      • et al.
      High tacrolimus intra-patient variability is associated with graft rejection, and de novo donor-specific antibodies occurrence after liver transplantation.
      ,
      • Contin M.
      • Alberghini L.
      • Candela C.
      • Benini G.
      • Riva R.
      Intrapatient variation in antiepileptic drug plasma concentration after generic substitution vs stable brand-name drug regimens.
      ].
      A high degree of variability has been described in response to ASD [
      • Contin M.
      • Alberghini L.
      • Candela C.
      • Benini G.
      • Riva R.
      Intrapatient variation in antiepileptic drug plasma concentration after generic substitution vs stable brand-name drug regimens.
      ,
      • Balestrini S.
      • Sisodiya S.M.
      Pharmacogenomics in epilepsy.
      ,
      • Johannessen Landmark C.
      • Johannessen S.I.
      • Tomson T.
      Host factors affecting antiepileptic drug delivery-pharmacokinetic variability.
      ]. Subjects with apparently similar types of epilepsy show different responses to the same ASD. Responsiveness and resistance to ASD were described in a pre-clinical model, along with resistance to multiple drugs [
      • Klein S.
      • Bankstahl M.
      • Loscher W.
      Inter-individual variation in the effect of antiepileptic drugs in the intrahippocampal kainate model of mesial temporal lobe epilepsy in mice.
      ]. DRE can be explained by mechanisms related to pharmacokinetics, pharmacodynamics, and pharmacogenomics in some patients with epilepsy. However, these fail to explain resistance in all patients.
      A large inter-individual variation in the concentration-to-dose ratio of perampanel was attributed to the fact that its metabolism is predisposed to interactions with enzyme-inducing ASD [
      • Yamamoto Y.
      • Usui N.
      • Nishida T.
      • et al.
      Therapeutic drug monitoring for perampanel in japanese epilepsy patients: influence of concomitant antiepileptic drugs.
      ]. In most pediatric patients with epilepsy treated with levetiracetam, there is no strong indication for a concentration-response association with efficacy or toxicity. It has been suggested that concentrations of levetiracetam do not correlate with the therapeutic value, implying that clinical judgment, without assessing drug concentrations, should be the main strategy [
      • Tan J.
      • Paquette V.
      • Levine M.
      • Ensom M.H.H.
      Levetiracetam Clinical Pharmacokinetic Monitoring in Pediatric Patients with Epilepsy.
      ]. The C3435 T polymorphism of the MDR1 gene has been proposed as a marker to predict drug-resistance in epilepsy [
      • Shaheen U.
      • Prasad D.K.
      • Sharma V.
      • et al.
      Significance of MDR1 gene polymorphism C3435T in predicting drug response in epilepsy.
      ]. Polymorphisms in CYP2D6, CYP2C9, CYP2C19, and CYP3A4 have been described in some patients with DRE [
      • Lopez-Garcia M.A.
      • Feria-Romero I.A.
      • Serrano H.
      • et al.
      Influence of genetic variants of CYP2D6, CYP2C9, CYP2C19 and CYP3A4 on antiepileptic drug metabolism in pediatric patients with refractory epilepsy.
      ]. SCN1A and SCN2A gene polymorphisms affect the response to VPA in some patients [
      • Shi L.
      • Zhu M.
      • Li H.
      • et al.
      SCN1A and SCN2A polymorphisms are associated with response to valproic acid in Chinese epilepsy patients.
      ]. Genetic variation in CACNA1G, CACNA1H, CACNA1I, and ABCB1 has been suggested to affect drug responsiveness [
      • Glauser T.A.
      • Holland K.
      • O’Brien V.P.
      • et al.
      Pharmacogenetics of antiepileptic drug efficacy in childhood absence epilepsy.
      ]. Genetic variability has been observed in the ABCB1 gene coding the efflux transporter multidrug resistance protein 1 (MDR1). However, population-based studies of ABCB1 polymorphisms differ in their conclusions [
      • Heinrich A.
      • Zhong X.B.
      • Rasmussen T.P.
      Variability in expression of the human MDR1 drug efflux transporter and genetic variation of the ABCB1 gene: implications for drug-resistant epilepsy.
      ].
      Intra-patient daily variability in ASD plasma levels in subjects stabilized with the same product over time has been described, with a several-fold variability in serum levels [
      • Contin M.
      • Alberghini L.
      • Candela C.
      • Benini G.
      • Riva R.
      Intrapatient variation in antiepileptic drug plasma concentration after generic substitution vs stable brand-name drug regimens.
      ]. The intra-subject variation in serum levels of levetiracetam (LEV), topiramate (TPM), and lamotrigine (LTG) following generic switch has been studied. An increase or decrease of a > 20 % alteration in drug concentration in the plasma was reported, while the patients’ received stable dosages, has been reported by therapeutic drug monitoring (TDM) studies. Both inter-day variability in intra-patient LEV, TPM, and LTG plasma levels were described, even in subjects stabilized with the same drug over a long period of time [
      • Contin M.
      • Alberghini L.
      • Candela C.
      • Benini G.
      • Riva R.
      Intrapatient variation in antiepileptic drug plasma concentration after generic substitution vs stable brand-name drug regimens.
      ].
      Taken together, the data suggest a high degree of variability in seizures and response to ASD, which can also partly explain the high rate of resistance that develops in these patients.

      1.6 A stepwise approach for establishing a platform for overcoming drug resistance in epilepsy

      Many biological systems are implementing variability patterns in their function as part of a process to identify the optimal response to different triggers. An “optimal state of variability” has been proposed to take on a ‘U shape’ representing chaotic variability in a steady state and complete predictability [
      • Ilan Y.
      Advanced tailored randomness: a novel approach for improving the efficacy of biological systems.
      ,
      • Ilan Y.
      Beta-glycosphingolipids as mediators of both inflammation and immune tolerance: a manifestation of randomness in biological systems.
      ,
      • Ilan Y.
      Randomness in microtubule dynamics: an error that requires correction or an inherent plasticity required for normal cellular function?.
      ,
      • Ilan Y.
      Generating randomness: making the most out of disordering a false order into a real one.
      ,
      • Stergiou N.
      • Decker L.M.
      Human movement variability, nonlinear dynamics, and pathology: is there a connection?.
      ,
      • Ilan Y.
      Why targeting the microbiome is not so successful: can randomness overcome the adaptation that occurs following gut manipulation?.
      ]. Regular dosing regimens have been suggested to be incompatible with physiological variability and to further add to the development of drug-resistance.
      Drug holidays are used for overcoming the loss of effect of chronic medications [
      • Weiner W.J.
      • Koller W.C.
      • Perlik S.
      • Nausieda P.A.
      • Klawans H.L.
      Drug holiday and management of Parkinson disease.
      ]. Anti-TNF re-induction subsequent to a drug holiday can overcome the loss of response [
      • Khanna R.
      • Levesque B.G.
      • Sandborn W.J.
      • Feagan B.G.
      Therapeutic drug monitoring of TNF antagonists in inflammatory bowel disease.
      ]. Intermittent dosing can recover clinical benefits, while reducing drug exposure and adverse effects. An intermittent dosing regimen for the anti-seizure effect of rapamycin, an mTOR inhibitor, was studied in a tuberous sclerosis complex (TSC) mouse model. Intermittent dosing of rapamycin using drug holidays of > 3 weeks preserves a substantial anti-seizure effect [
      • Rensing N.
      • Han L.
      • Wong M.
      Intermittent dosing of rapamycin maintains antiepileptogenic effects in a mouse model of tuberous sclerosis complex.
      ]. Dose modifications and introducing variability into anti-TNF therapies improves the response towards these agents [
      • De Luna-Preitschopf A.
      • Zwickl H.
      • Nehrer S.
      • Hengstschlager M.
      • Mikula M.
      Rapamycin maintains the chondrocytic phenotype and interferes with inflammatory cytokine induced processes.
      ]. In a prospective trial of patients with inflammatory bowel disease receiving biological therapy, loss of clinical response was noted in 36 % of patients on regular regimens vs. only 13 % of patients who were on a dose alteration regimen, irrespective of the lack of change in drug serum level [
      • Strik A.B.
      • Mould S.
      • Mathôt D.
      • Ponsioen R.
      • van den Brande C.
      • Jansen J.
      • et al.
      Dashboard driven vs. conventional dosing of infliximab in inflammatory bowel disease patients: the PRECISION trial.
      ].
      A stepwise approach is used for developing a platform for improving the effect of ASD [
      • Gelman R.
      • Bayatra A.
      • Kessler A.
      • Schwartz A.
      • Ilan Y.
      Targeting SARS-CoV-2 receptors as a means for reducing infectivity and improving antiviral and immune response: an algorithm-based method for overcoming resistance to antiviral agents.
      ,
      • Ilan Y.
      Order through disorder: the characteristic variability of systems.
      ,
      • Ilan Y.
      Advanced tailored randomness: a novel approach for improving the efficacy of biological systems.
      ]. Version 1.0 implements a random-number generator for introducing variability in dosing and time drug administration. This is done within the therapeutic window of the drug, or multiple drugs, that the patient is using. The introduction of “simple” variability is expected to overcome drug-resistance and improve the response to the medications that the patient is currently taking. Version 2.0, implements a closed-loop algorithm where inputs are based on clinically meaningful endpoints for generating therapeutic regimens. For, version 3.0, host and disease-related patterns of variability are quantified in a personalized manner, and then implemented into a true-random number generator. This version involves a process of continuous adaptation of algorithm output to inputs from quantifiable variability parameters. These include parameters associated with disease pathogenesis, host response, and the mechanism of action of the drug [
      • Gelman R.
      • Bayatra A.
      • Kessler A.
      • Schwartz A.
      • Ilan Y.
      Targeting SARS-CoV-2 receptors as a means for reducing infectivity and improving antiviral and immune response: an algorithm-based method for overcoming resistance to antiviral agents.
      ].
      The polyetiologic nature of epilepsy requires personalization of the therapy. Successful therapy design requires considering the effects of inherent variabilities in an individualized way [
      • Toni T.
      • Tidor B.
      Combined model of intrinsic and extrinsic variability for computational network design with application to synthetic biology.
      ]. In the second stage, closed-loop machine learning is implemented into the algorithm along with the introduction of several epilepsy-related ANS and chronobiology parameters. The large interdependency between multiple network properties in these systems, many of which behave randomly, is isolated and quantified. These personalized signatures are used as a means for implementing variability patterns in an individualized way. Epilepsy is characterized by periodic dynamics that augment the likelihood of seizures at certain times of the day, and which are vastly patient-specific. Predictive models for epilepsy are improved by circadian information [
      • Karoly P.J.
      • Ung H.
      • Grayden D.B.
      • et al.
      The circadian profile of epilepsy improves seizure forecasting.
      ]. Deep phenotyping, EEG-based studies of network dysfunction, quantifiable brain imaging, innate immunity processes, microRNA as therapeutic targets, and genetic variants have been proposed as tools for improving personalized therapy in epilepsy and can be implemented into the algorithm [
      • Rosenow F.
      • van Alphen N.
      • Becker A.
      • et al.
      Personalized translational epilepsy research - novel approaches and future perspectives: part I: clinical and network analysis approaches.
      ].
      Fig. 1 presents a schematic presentation of a closed-loop system into which multiple epilepsy parameters are introduced and a personalized tailored treatment is offered to improve the effect of ASD while reducing toxicity.
      Fig. 1
      Fig. 1A: The reciprocal relationship between drug resistant epilepsy, the autonomic nervous system, chronobiology, and chronotherapy. B: Schematic representation of an algorithm, which is based on implementation of epilepsy-related autonomic nervous system (ANS) and chronobiology parameters, along with variability patterns, is implemented in an individualized and dynamic way. ANS – autonomic nervous system; DRE – drug resistant epilepsy; HRV – heart rate variability; IGF1 – insulin-like growth factor 1; BDNF - brain derived neurotrophic factor; EEG – electroencephalogram; MEQ – morningness-eveningness questionnaire.
      In summary, DRE is a multifactorial process, affecting up to a third of all subjects with epilepsy. The ANS and chronobiology may contribute to the pathogenesis of the disease. A platform consisting of a closed-loop system, in which parameters based on ANS, chronotherapy, and variability are implemented in a personalized way, is being developed with an aim of improving the response to current medications, while reducing toxicity. Ongoing trials (NCT03843697; NCT03747705) have been designed to assess the implementation of these algorithms in patients and will provide insight into various underlying mechanisms, enabling the generation of improved treatment modalities. Additional studies are required for assessing the role of chronobiology in DRE and the potential for improving the response to anti-seizure medications using chronotherapy.

      Declaration of Competing Interest

      The authors declare no conflict of interest.

      Acknowledgement

      YI is the founder of Oberon Sciences.

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