The SANAD II study of the effectiveness and cost-effectiveness of levetiracetam, zonisamide, or lamotrigine for newly diagnosed focal epilepsy: an open-label, non-inferiority, multicentre, phase 4, randomised controlled trial

Background: Levetiracetam and zonisamide are licensed as monotherapy for patients with focal epilepsy, but there is uncertainty as to whether they should be recommended as first-line treatments because of insufficient evidence of clinical effectiveness and cost-effectiveness. We aimed to assess the long-term clinical effectiveness and cost-effectiveness of levetiracetam and zonisamide compared with lamotrigine in people with newly diagnosed focal epilepsy. Methods: This randomised, open-label, controlled trial compared levetiracetam and zonisamide with lamotrigine as first-line treatment for patients with newly diagnosed focal epilepsy. Adult and paediatric neurology services across the UK recruited participants aged 5 years or older (with no upper age limit) with two or more unprovoked focal seizures. Participants were randomly allocated (1:1:1) using a minimisation programme with a random element utilising factor to receive lamotrigine, levetiracetam, or zonisamide. Participants and investigators were not masked and were aware of treatment allocation. SANAD II was designed to assess non-inferiority of both levetiracetam and zonisamide to lamotrigine for the primary outcome of time to 12-month remission. Anti-seizure medications were taken orally and for participants aged 12 years or older the initial advised maintenance doses were lamotrigine 50 mg (morning) and 100 mg (evening), levetiracetam 500 mg twice per day, and zonisamide 100 mg twice per day. For children aged between 5 and 12 years the initial daily maintenance doses advised were lamotrigine 1·5 mg/kg twice per day, levetiracetam 20 mg/kg twice per day, and zonisamide 2·5 mg/kg twice per day. All participants were included in the intention-to-treat (ITT) analysis. The per-protocol (PP) analysis excluded participants with major protocol deviations and those who were subsequently diagnosed as not having epilepsy. Safety analysis included all participants who received one dose of any study drug. The non-inferiority limit was a hazard ratio (HR) of 1·329, which equates to an absolute difference of 10%. A HR greater than 1 indicated that an event was more likely on lamotrigine. The trial is registered with the ISRCTN registry, 30294119 (EudraCt number: 2012-001884-64). Findings: 990 participants were recruited between May 2, 2013, and June 20, 2017, and followed up for a further 2 years. Patients were randomly assigned to receive lamotrigine (n=330), levetiracetam (n=332), or zonisamide (n=328). The ITT analysis included all participants and the PP analysis included 324 participants randomly assigned to lamotrigine, 320 participants randomly assigned to levetiracetam, and 315 participants randomly assigned to zonisamide. Levetiracetam did not meet the criteria for non-inferiority in the ITT analysis of time to 12-month remission versus lamotrigine (HR 1·18; 97·5% CI 0·95–1·47) but zonisamide did meet the criteria for non-inferiority in the ITT analysis versus lamotrigine (1·03; 0·83–1·28). The PP analysis showed that 12-month remission was superior with lamotrigine than both levetiracetam (HR 1·32 [97·5% CI 1·05 to 1·66]) and zonisamide (HR 1·37 [1·08–1·73]). There were 37 deaths during the trial. Adverse reactions were reported by 108 (33%) participants who started lamotrigine, 144 (44%) participants who started levetiracetam, and 146 (45%) participants who started zonisamide. Lamotrigine was superior in the cost-utility analysis, with a higher net health benefit of 1·403 QALYs (97·5% central range 1·319–1·458) compared with 1·222 (1·110–1·283) for levetiracetam and 1·232 (1·112, 1·307) for zonisamide at a cost-effectiveness threshold of £20 000 per QALY. Cost-effectiveness was based on differences between treatment groups in costs and QALYs. Interpretation: These findings do not support the use of levetiracetam or zonisamide as first-line treatments for patients with focal epilepsy. Lamotrigine should remain a first-line treatment for patients with focal epilepsy and should be the standard treatment in future trials. Funding: National Institute for Health Research Health Technology Assessment programme

The SANAD II study of the effectiveness and cost-effectiveness of valproate versus levetiracetam for newly diagnosed generalised and unclassifiable epilepsy: an open-label, non-inferiority, multicentre, phase 4, randomised controlled trial

Background: Valproate is a first-line treatment for patients with newly diagnosed idiopathic generalised or difficult to classify epilepsy, but not for women of child-bearing potential because of teratogenicity. Levetiracetam is increasingly prescribed for these patient populations despite scarcity of evidence of clinical effectiveness or cost-effectiveness. We aimed to compare the long-term clinical effectiveness and cost-effectiveness of levetiracetam compared with valproate in participants with newly diagnosed generalised or unclassifiable epilepsy. Methods: We did an open-label, randomised controlled trial to compare levetiracetam with valproate as first-line treatment for patients with generalised or unclassified epilepsy. Adult and paediatric neurology services (69 centres overall) across the UK recruited participants aged 5 years or older (with no upper age limit) with two or more unprovoked generalised or unclassifiable seizures. Participants were randomly allocated (1:1) to receive either levetiracetam or valproate, using a minimisation programme with a random element utilising factors. Participants and investigators were aware of treatment allocation. For participants aged 12 years or older, the initial advised maintenance doses were 500 mg twice per day for levetiracetam and valproate, and for children aged 5–12 years, the initial daily maintenance doses advised were 25 mg/kg for valproate and 40 mg/kg for levetiracetam. All drugs were administered orally. SANAD II was designed to assess the non-inferiority of levetiracetam compared with valproate for the primary outcome time to 12-month remission. The non-inferiority limit was a hazard ratio (HR) of 1·314, which equates to an absolute difference of 10%. A HR greater than 1 indicated that an event was more likely on valproate. All participants were included in the intention-to-treat (ITT) analysis. Per-protocol (PP) analyses excluded participants with major protocol deviations and those who were subsequently diagnosed as not having epilepsy. Safety analyses included all participants who received one dose of any study drug. This trial is registered with the ISRCTN registry, 30294119 (EudraCt number: 2012-001884-64). Findings: 520 participants were recruited between April 30, 2013, and Aug 2, 2016, and followed up for a further 2 years. 260 participants were randomly allocated to receive levetiracetam and 260 participants to receive valproate. The ITT analysis included all participants and the PP analysis included 255 participants randomly allocated to valproate and 254 randomly allocated to levetiracetam. Median age of participants was 13·9 years (range 5·0–94·4), 65% were male and 35% were female, 397 participants had generalised epilepsy, and 123 unclassified epilepsy. Levetiracetam did not meet the criteria for non-inferiority in the ITT analysis of time to 12-month remission (HR 1·19 [95% CI 0·96–1·47]); non-inferiority margin 1·314. The PP analysis showed that the 12-month remission was superior with valproate than with levetiracetam. There were two deaths, one in each group, that were unrelated to trial treatments. Adverse reactions were reported by 96 (37%) participants randomly assigned to valproate and 107 (42%) participants randomly assigned to levetiracetam. Levetiracetam was dominated by valproate in the cost-utility analysis, with a negative incremental net health benefit of −0·040 (95% central range −0·175 to 0·037) and a probability of 0·17 of being cost-effectiveness at a threshold of £20 000 per quality-adjusted life-year. Cost-effectiveness was based on differences between treatment groups in costs and quality-adjusted life-years. Interpretation: Compared with valproate, levetiracetam was found to be neither clinically effective nor cost-effective. For girls and women of child-bearing potential, these results inform discussions about benefit and harm of avoiding valproate. Funding: National Institute for Health Research Health Technology Assessment Programme

Endoplasmic Reticulum-Plasma Membrane Junctions: Novel Sites For Phosphoinositide Regulation

Endoplasmic reticulum-plasma membrane (ER-PM) contact sites are specialized stable regions of the ER that are tightly apposed to the PM. These conserved organelle junctions are thought to function as rapid and direct avenues for inter-organelle communication between the ER and PM. However, a major obstacle in the study of ER-PM contacts has been the identification of the factors that form and stabilize these structures. By trying to understand how Sac1, an ER anchored phosphoinositide phosphatase, regulates the phosphoinositide lipid phosphatidylinositol-4-phosphate (PI4P) on the PM, we have gained critical insight into ER-PM contact site formation and function. Here I present work that demonstrates Sac1 functions at ER-PM contacts to dephosphorylate PI4P on the PM. In the first part of my thesis, I uncovered a role for the yeast oxysterol binding protein homolog (Osh) family as critical regulators of PI4P metabolism. The Osh proteins are known PI4P effectors that localize to ER-organelle contact sites, including ERPM. We found that the entire Osh family regulates PI4P metabolism in vivo, through Sac1, and the Osh proteins stimulate Sac1 phosphatase activity in vitro. We propose that the Osh proteins serve as sensors of PI4P and activators of Sac1 at ER-PM contacts. In the second part of my thesis, I identified the tricalbin proteins (orthologs of the extended synaptotagmins), Ist2 (a member of the TMEM16 family of ion channels), and the yeast VAP proteins, Scs2/22 as ERPM tethering proteins. Strikingly, cells lacking all three families of tethering proteins display a dramatic retraction of the ER from the PM and an accumulation of cytoplasmic ER structures. These mutants also exhibit large accumulation of PI4P on the PM, consistent with a critical role for ER-PM contacts in regulating Sac1 function. Importantly, the generation of a strain lacking ER-PM junctions allowed us the opportunity to investigate novel functions for these structures. In addition to the defect in PI4P turnover, the unfolded protein response (UPR) was constitutively activated in cells lacking ER-PM junctions. This work provides a molecular mechanism for ER-PM contact site formation and identifies PI4P metabolism and ER homeostasis as new ER-PM contact site functions

The clinical patterns of frontal lobe epilepsy

The aims of this study were to define patterns of seizure semiology aid see if these can be localised to particular regions, with special emphasis on frontal lobe epilepsies; to establish clinical features differentiating frontal and temporal lobe epilepsies; to assess current classification of frontal lobe epilepsies and to analyse partial epilepsies in a general population. The method; used differ from other studies in this area, especially with respect to breadth of case mix; delineation of clinical syndromes, using statistical techniques and prospective analysis of investigative abnormalities in relation to clinically defined seizure types. Patients were selected from hospital records with evidence of partial seizure onset, on the basis of focal imaging abnormality, interictal or ictal EEG abnormality and clinical seizure pattern. Two hundred and fifty-two patients with 352 seizure types were identified. Clinical seizure manifestations were recorded prospectively and encoded according to sequential occurrence during she seizure. These data were entered into a statistical cluster analysis, which was refined to 14 groupings, each corresponding to a different seizure type. These patterns were displayed in flow charts, allowing assessment of seizure evolution. Interictal EEG spike distribution and ictal EEG onset were related to each seizure type. The 126 lesions identified on neuroimaging were measured by a template technique, aid related to each seizure type, using chi-square analysis. Investigations were also related to each other and the clinical seizure associations of pure frontal and pure temporal lesions were assessed. The database of the National General Practice Study of Epilepsy (NGPSE) was also analysed, using a clinical classification, to determine the relative frequency of different partial seizure types in a general population. Of 14 clinical seizure groupings, 2 had associations with frontal regions and 2 perirolandic associations. Focal clonic and somatosensory seizures were associated with perirolandic lesions and other sensory modalities and experiential phenomena with temporal EEG and imaging abnormalities. Seizures characterised by tonic posturing were strongly associated with lesions of the lateral premotor cortex and frontocentral EEG abnormalities. Seizures characterised by bizarre oi frenetic motor activity "motor agitation" were associated with frontal EEG abnormalities and with lesions of the frontopolar or orbitofrontal cortex. These latter seizures were commonly nocturnal and occurred with higher frequency than those in other groups but no other consistent pattern in diurnal variation or seizure frequency emerged. For all seizure types with statistically significant associations with specific lesion sites, there was a substantial minority of cases associated with lesions at different site. A direct comparison of seizures associated with pure frontal and pure temporal lesions confirmed earl) version, clonic activity and tonic posturing as frontal characteristics but found no consistent differences in characteristics of seizure timing. Seizures with startle sensitivity occurred in 19 cases; MRI suggested lesions in the lateral frontal region or the perisylvian region in this group. The NGPSE data supported a high frequency of seizure types associated with frontal lobe abnormalities in the general population and a good prognosis of all partial seizure categories. These findings lend some support to the localisation of seizure types attributed to orbitofrontal and temporal lobes by the ILAE but suggests that many regions of the frontal lobes do not have specific associated seizure types, and that many seizure types, although being associated with one cerebral region, may relatively frequently be due to lesions in other sites. The predictive value of clinical seizure type is thus less than described from retrospective and highly selected post-surgical series, on which the classification is based. The hospital and population-based studies suggest that seizure frequency, timing and prognosis are more related to the population under study than the anatomical site of the underlying lesion

Ubiquitin-Dependent Regulation of Stem Cell Biology

The growth of a metazoan body relies on a series of highly coordinated cell-fate decisions by stem cells which can undergo self-renewal, reversibly enter a quiescent state, or terminally commit to a cell specification program. To guide their decisions, stem cells make frequent use of ubiquitylation, a post-translational modification that can affect the activity, interaction landscape, or stability of stem cell proteins. In this review we discuss novel findings that have provided insight into ubiquitin-dependent mechanisms of stem cell control and revealed how an essential and highly conserved protein modification can shape metazoan development

Discovery of a Covalent FEM1B Recruiter for Targeted Protein Degradation Applications

Proteolysis Targeting Chimeras (PROTACs), heterobifunctional compounds that consist of protein-targeting ligands linked to an E3 ligase recruiter, have arisen as a powerful therapeutic modality for targeted protein degradation (TPD). Despite the popularity of TPD approaches in drug discovery, only a small number E3 ligase recruiters are available for the >600 E3 ligases that exist in human cells. Here, we have discovered a cysteine-reactive covalent ligand EN106 that targets FEM1B, an E3 ligase recently discovered to be critical in cellular reductive stress response. EN106, through targeting cysteine C186, disrupts FEM1B substrate recognition of FNIP1. We further establish that EN106 can be used as a covalent recruiter for FEM1B in TPD applications, in which we demonstrate that a PROTAC linking EN106 to the BET Bromodomain inhibitor JQ1 leads to the selective NEDDylation, proteasome, and FEM1B-dependent degradation of BRD4 in cells. Our study showcases a covalent ligand that targets a substrate recognition site within the E3 ligase FEM1B involved in reductive stress response and highlights the utility of covalent ligand screening in expanding the arsenal of E3 ligase recruiters that can be deployed for TPD applications

Structural basis and regulation of the reductive stress response.

Although oxidative phosphorylation is best known for producing ATP, it also yields reactive oxygen species (ROS) as invariant byproducts. Depletion of ROS below their physiological levels, a phenomenon known as reductive stress, impedes cellular signaling and has been linked to cancer, diabetes, and cardiomyopathy. Cells alleviate reductive stress by ubiquitylating and degrading the mitochondrial gatekeeper FNIP1, yet it is unknown how the responsible E3 ligase CUL2FEM1B can bind its target based on redox state and how this is adjusted to changing cellular environments. Here, we show that CUL2FEM1B relies on zinc as a molecular glue to selectively recruit reduced FNIP1 during reductive stress. FNIP1 ubiquitylation is gated by pseudosubstrate inhibitors of the BEX family, which prevent premature FNIP1 degradation to protect cells from unwarranted ROS accumulation. FEM1B gain-of-function mutation and BEX deletion elicit similar developmental syndromes, showing that the zinc-dependent reductive stress response must be tightly regulated to maintain cellular and organismal homeostasis