finbar o’callaghan reader in paediatric neuroscience · finbar o’callaghan reader in paediatric...

UCL - Institute of Child Health

Medical treatments – where are we?

Finbar O’Callaghan Reader in Paediatric Neuroscience


Plan of the talk •  Should we treat? •  What to treat with? •  Difficulties in evaluating treatment? •  Looking at the evidence

–  Infantile Spasms –  Ohtahara –  MPSI –  TSC –  Dravet’s

•  Possible new avenues •  Conclusions


Should we always be treating?

•  James Edwin West •  Born in 1840 in Tonbridge to

William James West and Mary Dashwood

•  Infantile spasms developed at 8 months

•  Developmental regression •  Spasms stopped at 3 years •  Ongoing epilepsy and learning

difficulty

•  Leeches •  Cold applications to head •  Calomel purgatives •  Lancing of gums •  Warm baths •  Castor oil •  Hydrocyanic acid


Should we always be treating?

•  Treatment may be relatively ineffective

•  Treatment may be harmful (? GABA agonists in infancy)

•  Very difficult to “sit on our hands”


•  5 categories of lead time:

•  < 7 days •  8-14 days •  15- 28 days •  1-2 months •  > 2 months

•  Each category in lead time to treatment lead to reduction in DQ of 3.9 points (95% CI 0.4 to 7.5 p=0.014)


“Infantile spasms should be treated with nitrazepam and a month of corticotrophin followed by 2 months of prednisolone unless the year ends with an odd number, when sodium valproate and prednisolone are de riguer, although you could make do with vigabatrin and 3 weeks of hydrocortisone if your favourite colour is blue and Jupiter is about to collide with Mars.” “Treatment of infantile spasms”

Arch Dis Child 1995:73;188.


Difficulties in evaluating treatments in infantile epilepsy

•  Double-blind RCT is thought to be “gold standard for evaluating treatments.

•  Rarity of conditions –  Lack of power –  Expense

•  Blinding often difficult


What outcomes are important?

•  Reduction in spasms •  Abolition •  Electro-clinical •  Development

–  At what stage?

•  Future epilepsy


Infantile spasms


Infantile Spasms

•  18 RCTs •  12 different pharmacological treatments

–  Vigabatrin –  ACTH (9 different regimens and preparations) –  Prednisolone –  Prednisone –  Hydrocortisone –  Nitrazepam –  Sodium Valproate –  Sulthiame –  Ganoxolone –  Methysergide –  Alpha-Methylparatyrosine –  Magnesium Sulphate


Quality of studies

•  Methodology –  Only two studies had >

70 participants –  6 out of 18 stated

method of randomisation

–  4 reported concealment of allocation

–  In 6 studies assessors were blinded to allocation


Trials of agent versus placebo

•  Vigabatrin versus placebo (Appleton et al, 1999) –  40 participants –  7/20 on VGB spasm free vs 2/20 on placebo

•  Sodium valproate versus placebo (Dyken 1985) –  Significant reduction in “spasm index” on valproate

versus placebo •  Sulthiame versus placebo (Debus 2004)

–  8/23 on sulthiame spasms free versus 1/23 on placebo


Low-dose versus high dose vigabatrin

•  High dose regime (100-148 mg/kg/day) •  Low dose (18-36 mg/kg/day) •  17/107 on high dose versus 8/114 on low dose

became spasm free (p = 0.04)


Vigabatrin versus hormonal treatment (ACTH, high-dose prednisolone, tetracosactide)

•  Vigevano et al. (1997) –  11/23 on VGB versus 14/19 on ACTH = spasm free

•  Askalan et al. (2003) –  6/6 on VGB versus 3/3 on ACTH = spasm free

•  Lux et al. (2004) –  28/52 on VGB versus 40/55 on hormonal Rx = spasm free

•  Combining results: –  45/81 on VGB versus 57/77 on hormonal Rx = spasm free (Peto OR

0.42 95% CI 0.21 to 0.8)


ACTH versus high dose prednisolone

•  Nested within UKISS study •  Comparison of ACTH 40-60 Units alt die vs

Prednisolone 40-60 mg/day

•  19/25 on ACTH versus 21/30 became spasm free (p = n.s.)


ACTH versus low-dose prednisone

•  Hrachovy et al. (1983), Baram et al. (1996) •  Prednisone has to be metabolised to prednisolone •  Hrachovy

–  ACTH (20 – 30 units/day) vs Prednisone (2mg/kg/day) –  ACTH (5/12 spasm free) vs Prednisone (4/12 spasm

free) •  Baram

–  ACTH (150 units/m2/day) vs Prednisone (2mg/kg/day) –  ACTH (13/15 spasm free) vs Prednisone (4/14 spasm

free)


High dose versus low dose ACTH

•  Hrachovy et al. (1994), Shu et al. (2009) •  Hrachovy

–  High dose (150 units/m2/day for 3 weeks) – 13/30 responded

–  Low dose (20-30 units/day for 2-6 weeks) – 14/29 responded

•  Shu –  High dose (50 IU/day for 3 weeks) – 53% responded –  Low dose (0.4-1.0 IU/kg/day for 2 weeks) – 60%

responded


Conclusions (Cochrane)

•  Hormonal treatments lead to faster resolution of spasms and in more infants than vigabatrin

•  Response without subsequent relapse may be no different

•  If prednisolone or vigabatrin used – use high dose •  Hormonal therapies may be associated with better

developmental outcome in those with no proven aetiology


Conclusions (American Academy of Neurology)

•  Low dose ACTH should be considered •  ACTH and VGB may be considered for short term

treatment of spasms (preference for ACTH) •  Hormonal treatment considered in preference to

VGB for “cryptogenic” spasms •  Shorter treatment lag with either hormonal therapy

or VGB may be associated with better developmental outcome


Ohtahara Syndrome •  Early onset (first month) •  Tonic spasms and focal

seizures •  S-B pattern on EEG •  Underlying structural

pathology •  Treatments

–  Keto diet –  Levetiracetam –  Zonisamide –  High dose phenobaritone –  Valproate


Migrating Partial Seizures of Infancy •  Infantile onset (< 6 mo) •  Almost continuous

migrating polymorphous seizures

•  Migrating multifocal discharges

•  Psychomotor deterioration •  Treatments tried:

–  Keto diet –  Levetiracetam –  Rufinamide –  Stiripentol –  Bromides McTague A et al. Brain 2013


Tuberous sclerosis complex

•  Comparison between vigabatrin (150 mg/kg/day) and hydrocortisone (15 mg/kg/day)

•  22 patients in trial •  11/11 on vigabatrin vs 5/11 on hydrocortisone

spasm free at 1 month (p < 0.01) •  ? Methodological problems


Dravet Syndrome


Ketogenic Diet

•  Consider if hormonal therapies and vigabatrin fail in spasms

•  Consider in malignant intractable epilepsies (e.g. Ohtahara, MPSI)

•  ? At least as effective as any new add-on anti-convulsant medication

•  Clinical trials


Therapies to be cautious about


New ways forward?

•  Prophylaxis •  Combination therapy •  mTOR inhibition •  Neuroprotection


Prophylaxis

•  Jozwiak et al. Epilepsia 2007 & EJPN 2011 •  Methodology

–  45 infants: 31 received standard therapy and 14 received prophylactic therapy –  Significant reduction in LD in prophylactic group at 2 years (48% vs 14% p = 0.031) –  Significant higher proportion seizure free in prophylactic group (93% vs 35% p =

0.004)

•  Can we reliably identify children who will go on to develop epileptic encephalopathy?

•  Problems of giving treatment with known side-effects (e.g VFDs) to children who would never develop epilepsy

•  Potentially other adverse effects of treatment –  GABA agonists and the developing brain


Combination therapy

•  Some patients will respond to one therapy and not another

•  Rationale for –  Combination of VGB and

hormonal therapy vs hormonal therapy alone

•  Combine therapies – multiply risk of side-effects/damage


•  mTOR overactivation implicated in genetic forms of infantile spasms e.g. TSC, NF-1, PTEN

•  Also implicated in multiple-hit animal model of infantile spasms


Rapamycin suppresses axon sprouting by somatostatininterneurons in a mouse model of temporal lobe epilepsy

*yPaul S. Buckmaster and *Xiling Wen

Departments of *Comparative Medicine and yNeurology & Neurological Sciences, Stanford University, Stanford, California, U.S.A.

SUMMARY

Purpose: In temporal lobe epilepsy many somatostatininterneurons in the dentate gyrus die. However, somesurvive and sprout axon collaterals that form new syn-apses with granule cells. The functional consequences ofc-aminobutyric acid (GABA)ergic synaptic reorganizationare unclear. Development of new methods to suppressepilepsy-related interneuron axon sprouting might beuseful experimentally.Methods: Status epilepticus was induced by systemicpilocarpine treatment in green fluorescent protein(GFP)-expressing inhibitory nerurons (GIN) mice inwhich a subset of somatostatin interneurons expressesGFP. Beginning 24 h later, mice were treated with vehicleor rapamycin (3 mg/kg intraperitoneally) every day for2 months. Stereologic methods were then used to esti-mate numbers of GFP-positive hilar neurons per dentategyrus and total length of GFP-positive axon in the molecu-lar layer plus granule cell layer. GFP-positive axon densitywas calculated. The number of GFP-positive axon cross-ings of the granule cell layer was measured. Regressionanalyses were performed to test for correlations betweenGFP-positive axon length versus number of granule cellsand dentate gyrus volume.

Key Findings: After pilocarpine-induced status epilepti-cus, rapamycin- and vehicle-treated mice had approxi-mately half as many GFP-positive hilar neurons as didcontrol animals. Despite neuron loss, vehicle-treatedmice had over twice the GFP-positive axon length perdentate gyrus as controls, consistent with GABAergicaxon sprouting. In contrast, total GFP-positive axonlength was similar in rapamycin-treated mice and con-trols. GFP-positive axon length correlated most closelywith dentate gyrus volume.Significance: These findings suggest that rapamycinsuppressed axon sprouting by surviving somatostatin/GFP-positive interneurons after pilocarpine-induced sta-tus epilepticus in GIN mice. It is unclear whether the effectof rapamycin on axon length was on interneurons directlyor secondary, for example, by suppressing growth of gran-ule cell dendrites, which are synaptic targets of interneu-ron axons. The mammalian target of rapamycin (mTOR)signaling pathway might be a useful drug target for influ-encing GABAergic synaptic reorganization after epilepto-genic treatments, but additional side effects of rapamycintreatment must be considered carefully.KEY WORDS: mTOR, Synaptogenesis, GABA, Dentategyrus, Hippocampus.

Loss of somatostatin interneurons in the dentate gyrus iscommon in patients with temporal lobe epilepsy (de Lane-rolle et al., 1989; Sundstrom et al., 2001). Paradoxically, inthe same region, somatostatin-immunoreactive axons per-sist and appear exuberant, which suggests that survivinginterneurons sprout axons and form new synapses withgranule cells (Mathern et al., 1995). Support for this viewrecently came from a mouse model of temporal lobe epi-lepsy. In the molecular layer of the dentate gyrus, somato-statin-immunoreactivity of axons is weak in mice compared

to that of other species (Buckmaster et al., 1994). However,in GIN mice a subset of somatostatin interneurons expressenhanced green fluorescent protein (GFP), which is a supe-rior marker for axons (Oliva et al., 2000). Epileptic pilocar-pine-treated GIN mice display fewer hilar GFP-positiveneurons, consistent with loss of somatostatin interneurons,but greater GFP-immunoreactive axon length and more con-nectivity to granule cells, suggesting axon sprouting andsynaptogenesis (Zhang et al., 2009).

The functional consequences of epilepsy-related somato-statin axon sprouting are unclear. It might compensate forthe loss of some interneurons by restoring and strengtheningfeedback inhibition of granule cells. But there also areproepileptic alternatives. Possibilities include hypersyn-chronization of excitatory hippocampal neurons (Babbet al., 1989), generation of depolarizing c-aminobutyricacid (GABA) responses in principal cells (Staley et al.,

Accepted July 18, 2011; Early View publication August 29, 2011.Address correspondence to Paul Buckmaster, 300 Pasteur Drive, R321

Edwards Building, Department of Comparative Medicine, Stanford Univer-sity, Stanford, CA 94305, U.S.A. E-mail: [email protected]

Wiley Periodicals, Inc.ª 2011 International League Against Epilepsy

Epilepsia, 52(11):2057–2064, 2011doi: 10.1111/j.1528-1167.2011.03253.x

FULL-LENGTH ORIGINAL RESEARCH

2057

•  Somatostatin interneurons in dentate gyrus decrease in patients with TLE •  Surviving interneurons sprout axons ? Pro-epileptogenic • Rapamycin suppressed axon sprouting in a mouse model of TLE

surviving somatostatin/GFP-positive hilar interneuronssprout axons after pilocarpine-induced status epilepticus(Zhang et al., 2009). Absolute values of GFP-positiveaxon length are larger in the present study, probablybecause tissue was sectioned more thinly (see Methods),which facilitated immunostaining and axon reconstructionfor measurement. However, relative increases of pilo-carpine-treated versus control mice were similar in bothstudies (220% of controls in the present analysis; 192% ofcontrols in the previous study). Mice that experiencedstatus epilepticus and were treated with rapamycin for2 months had 37.2 € 2.2 m (range 30.6–43.1 m) of GFP-positive axon per molecular layer plus granule cell layer,which was not significantly different from controls(p = 0.48) but was less than that of vehicle-treated mice(p < 0.001). These findings suggest rapamycin suppressedaxon sprouting by GFP-positive interneurons after pilocar-pine-induced status epilepticus.

Changes in total axon length per dentate gyrus could beattributable to changes in axon density, changes in total vol-ume of the dentate gyrus, or both. To measure axon density,

the average length of GFP-positive axon in a volume oftissue 1 lm2 and the thickness of a section (40 lm) wascalculated. Average axon density was 0.76 € 0.04 in controlmice, 0.91 € 0.07 in mice that experienced statusepilepticus and were then treated with vehicle, and0.74 € 04 in mice that experienced status epilepticus andwere then treated with rapamycin (Fig. 3C). The average invehicle-treated mice was approximately 1.2 times that ofthe other groups, but the difference was not significant(p = 0.068, ANOVA). Average numbers of GFP-positiveaxon crossings of the granule cell layer in sections fromthe mid-septotemporal level were similar in control(0.11 € 0.01 crossings per 1-lm-length of granule celllayer), vehicle-treated (0.11 € 0.01 crossings), and rapamy-cin-treated mice (0.10 € 0.01 crossings) (Fig. 3D), despitefewer interneurons in mice that had experienced statusepilepticus.

The dentate gyrus enlarges after pilocarpine-inducedstatus epilepticus in mice, and this effect is suppressed byrapamycin (Zhang et al., 2009). Tissue utilized in thepresent study was collected as part of a previous study

A1 A2

B1 B2

C1 C2

Figure 1.GFP-immunostaining of the dentategyrus in a control mouse (A) and inmice that had experiencedpilocarpine-induced statusepilepticus and then were treateddaily for 2 months with vehicle (B)or 3 mg/kg rapamycin (C).Asterisks in left panels indicateareas shown at higher magnificationin right panels. m = molecularlayer, g = granule cell layer,h = hilus, CA3 = CA3 pyramidalcell layer.Epilepsia ILAE

2060

P. S. Buckmaster and X. Wen

Epilepsia, 52(11):2057–2064, 2011doi: 10.1111/j.1528-1167.2011.03253.x


•  mTOR inhibition may prevent essential repair of brain in the context of brain injury

•  Paradoxical effect of mTOR inhibitors in animal models of status epilepticus

•  Timing of administration

www.landesbioscience.com Cell Cycle 2281

Cell Cycle 9:12, 2281-2285; June 15, 2010; © 2010 Landes Bioscience EXTRA VIEW EXTRA VIEW

Key words: kainate, apoptosis, epilepsy, seizure, rat

Submitted: 03/22/10

Revised: 03/23/10

Accepted: 04/27/10

Previously published online: www.landesbioscience.com/journals/cc/article/11866*Correspondence to: Michael Wong; Email: [email protected]

Identification of cell signaling mechanisms mediating seizure-related

neuronal death and epileptogenesis is important for developing more effective therapies for epilepsy. The mammalian target of rapamycin (mTOR) pathway has recently been implicated in regulat-ing neuronal death and epileptogenesis in rodent models of epilepsy. In par-ticular, kainate-induced status epilep-ticus causes abnormal activation of the mTOR pathway, and the mTOR inhibi-tor, rapamycin, can decrease the devel-opment of neuronal death and chronic seizures in the kainate model. Here, we discuss the significance of these findings and extend them further by identifying upstream signaling pathways through which kainate status epilepticus activates the mTOR pathway and by demonstrat-ing limited situations where rapamycin may paradoxically increase mTOR acti-vation and worsen neuronal death in the kainate model. Thus, the regulation of seizure-induced neuronal death and epi-leptogenesis by mTOR is complex and may have dual, opposing effects depend-ing on the physiological and pathologi-cal context. Overall, these findings have important implications for designing potential neuroprotective and antiepi-leptogenic therapies that modulate the mTOR pathway.

Introduction

Epilepsy is one of the most common neurological disorders, affecting approxi-mately 1% of people, and is characterized

Regulation of cell death and epileptogenesis by the mammalian target of rapamycin (mTOR)A double-edged sword?

Ling-Hui Zeng,1 Sharon McDaniel,2 Nicholas R. Rensing2 and Michael Wong2

1Department of Pharmacy; Zhejiang University City College; Hangzhou, Zhejiang China; 2Department of Neurology and the Hope Center for Neurological

Disorders; Washington University School of Medicine; St. Louis, MO USA

by significant morbidity and mortality. Although there are a variety of underly-ing causes for epilepsy, seizures themselves are often implicated in causing progres-sive epileptogenesis and neuronal death, contributing to medically-intractable epi-lepsy and co-morbid cognitive deficits. Currently available medications simply suppress seizure symptomatically, but do not appear to prevent seizure-induced brain injury or reverse the underlying mechanisms of epileptogenesis.1 Thus, it is now widely recognized that novel thera-pies for epilepsy need to be developed that have neuroprotective, antiepileptogenic or disease-modifying properties.2-4

In order to develop disease-modifying therapies for epilepsy, a better under-standing of the cellular and molecu-lar mechanisms of epileptogenesis and seizure-induced brain injury is required. While traditionally seizure medications have targeted ion channels and neurotrans-mitters receptors that directly contribute to neuronal excitability, a recent trend has been to identify and target primary cell signaling pathways that initially trig-ger downstream mechanisms mediating neuronal injury and epileptogenesis. The mammalian target of rapamycin (mTOR) signaling pathway represents a ratio-nal candidate, because mTOR regulates numerous cellular functions and mecha-nisms that affect cell survival and death, neuronal excitability and epileptogenesis.5 Furthermore, available drugs exist that specifically inhibit mTOR and could be readily tested as neuroprotective and anti-epileptogenic therapies for epilepsy.

www.landesbioscience.com Cell Cycle 2283

also been implicated in mediating neuro-nal death and epileptogenesis in rodent models of acquired epilepsy due to brain injury. The mTOR pathway is activated in animal models of traumatic brain injury (TBI) and rapamycin has neuroprotective effects against neuronal death and func-tional deficits following TBI, although effects on posttraumatic epilepsy have not been described.27,28 mTOR is also trig-gered in the pilocarpine model of acquired epilepsy and mediates axonal sprouting, a putative mechanism of epileptogenesis.29 In a recent study, we have reported that the mTOR pathway is involved in neu-ronal death and epileptogenesis in the related kainate model of limbic epilepsy in rats.30 In the kainate model, an initial episode of prolonged seizures (status epi-lepticus), induced by administration of the glutamate agonist, kainate, triggers neuronal death and other cellular and molecular changes that promote epilep-togenesis. After recovery from the status epilepticus and following a latent period of days to weeks, these changes lead to the development of spontaneous seizures. In this model, we showed that kainate causes activation of the mTOR pathway both acutely during status epilepticus and more chronically for several weeks coinciding with the latent period of epileptogenesis.30 The mTOR inhibitor rapamycin prevents the abnormal kainate-induced mTOR activation and, depending on the timing of the rapamycin administration, causes a variable decrease in putative cellular mechanisms of epileptogenesis, includ-ing hippocampal neuronal death, neuro-genesis and axonal sprouting. Rapamycin also causes a corresponding decrease in the development of spontaneous seizures.30 Thus, these studies suggested that mTOR plays a critical role in activating multiple downstream mechanisms of neuronal injury and epileptogenesis in the kainate model and that rapamycin has neuropro-tective and antiepileptogenic actions in this model.

Figure 2. Kainate status epilepticus activates Akt, an upstream regulator of the mTOR pathway. Western blots of hippocampal homogenates were performed at various time points following kainate status epilepticus in adult rats. Compared to control rats, the ratio of P-Akt to total Akt was increased acutely with kainate status epilepticus and remained elevated for several weeks. *p < 0.05, **p < 0.01 by ANOVA, compared to the control group.

Figure 3. Rapamycin causes paradoxical exacerbation of kainate-induced mTOR activation when administered within one hour of kainate. Adult rats were injected with vehicle (Con), kainate (15 mg/kg, i.p.), or rapamycin (6 mg/kg) at different intervals before or after kainate. Kainate alone (KA) causes increased mTOR activation, as reflected by the ratio of phospho-S6 to total S6 expression measured 7 days after kainate injection, compared to vehicle (Con). Pretreatment with rapamycin one day prior to kainate inhibits the kainate-induced mTOR activation (Pre-1d). In con-trast, rapamycin administered within one hour before (Pre-1 h) or after (Post-1 h) kainate causes a paradoxical increase in the kainate-induced mTOR activation. *p < 0.05, ***p < 0.001 by ANOVA, compared to the KA group.


Neuroprotection?

•  Vigabatrin vs Vigabatrin + Flunarizine –  Non-significant difference in spasm cessation –  Non-significant difference in development @ 24 months –  BUT – significant difference in development in group

with no known aetiology (84.1 vs 72.3 p = 0.03)


Conclusions •  Evidence base is poor but slowly improving •  Treat early… but •  Beware of polypharmacy and side-effects •  Beware of using drugs that make the situation worse

–  e.g. Carbamazepine

•  Infantile spasms –  Hormonal therapy or vigabatrin –  Hormonal therapy controls spasms faster and may be associated

with better cognitive outcome in no-cause found group •  Some evidence for syndrome/disease specific treatments

–  e.g. Dravet, TSC

•  Ketogenic diet •  Promising new avenues

–  e.g. mTOR inhibition, Combination therapy

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