Workshop Models for proconvulsant drugs and precautions for entry into man Jean-Pierre MARTINOLLE

1. Workshop Models for proconvulsant drugs and precautions for entry into man Jean-Pierre MARTINOLLE Safety Pharmacology Advisory Group

2. SEIZURE / CONVULSIONFacts … • Nervous systems AEs responsible in 22% of cases for trial halted, delayed, development stopped, 39% of labelling restriction and for 25% of withdrawal from sales (2010) • Nervous systems AEs range from • High concern: seizure liability, neuropathy, brain phospholipidosis, abuse liability, ocular toxicity, suicidality • Medium concern: sedation/sleep disorders, motor/coordination effects, cognition, dizziness, auditory, psychiatric, somatosensory, anxiety… • Low concern: headache, autonomic, appetite, nausea… Thundiyil et al, J Med Toxicol 2011 Adapted from Redfern WS et al. SOT 2010 Poster 1081 DrugCite 2013

3. SEIZURE / CONVULSIONDefinition / Risk Unknown • SEIZURE • abnormal brain electrical discharge, possibly leading to sudden, involuntary, time-limited alterations in behavior, motor activity, autonomic function, consciousness or sensation • recorded through EEG • CONVULSION • a violent and involuntary muscular activity (as a consequence of brain seizure) of one or several muscles which are part of an organ, or of limbs, or generalized to all the body • detected through OBSERVATION Known Structural class SeizureSensitivity Undetected PreviousSeizure Off Target Pharmacological class History On Target Disease History Population related Facilitator Compound related CNS activity Directconvulsant SEIZURE RISK Healthy Patient BBB penetration PgpMRP, BCRP Chemicalproperties Indication Co-disease Administration related Co-medication Brain concentration Route Cmax Single dose Acute Seizure Metabolite Repeated dose Chronic Seizure • DRUG-INDUCED SEIZURE • 1% (world) with epilepsy, 3-10% (world ) will experience seizures in their life10% people with natural seizure tendency if a stimulus (lower threshold)Everyone could have a seizure (particular set of circumstances) • 1.2% of seizures at emergency department6% of new seizures are directly drug-related, 9% of status epilepticus are directly drug-related2% of mortality when it occurs, 5% of further status epilepticus when it occurs Kindling

4. Preclinical Predictivity • Seizureisundetectablewithoutsophisticatedmethodslike EEG or bio-imaging • Convulsionistricky to detect if onlybased on observation • Rare event • Rarelyoccuring in all speciestested • Oftenresolvingquicklythus not seen (if limited time of observation) • Not alwaysleading to deaththus no alert Then chance of detectiongenerallyincreaseswith the muliplication of studies and species but can lead to a latediscovery of the risk !

5. What are the chances to detect seizure / convulsion during preclinical ? No chance to detect a seizure / Low chance to observe a convulsion if no dedicated study

6. SEIZURE / CONVULSIONImproving Preclinical Risk assessment • Target • specific localisation in the brain • specific CNS role in particular in relation to seizure • Drug class • specific CNS activity • any compound on the market, anywithdrawal • Drug structure • use of SAR approach, in silico tools • Target population • Healthy volunteers (undetected seizure sensitivity, unknown history of seizure) • Patients (undetected seizure sensitivity, unknown history of seizure, specific disease history, targeted pathology, BBB altered, poly-medication, age (elderly or <5 years)) • Brainpenetration • Secondarypharmacology

7. strong evidence evidence unclear evidence SEIZURE / CONVULSIONSecondary pharmacology black list

8. 100 80 60 Relative Current (%) 40 1.00 20 0.80 0.60 100 0.01 0.1 1 10 I/IMax Pentylenetetrazole (mM) 0.40 0.20 0.1 1 10 GABA (µM) SEIZURE/CONVULSION LIABILITYGABAA receptor patch-clamp • GABA A RECEPTOR PATCH-CLAMP (electrophysiology) is aimed at quantitatively determinate the property and potency of the tested compound on GABA A receptor (ligand-gated Cl- channel). Many convulsants modulate GABA A receptor and inhibition of GABA A transmission induces convulsions • Results: agonist, antagonist, modulator, EC50, IC50 • Pros • High scientific reliability demonstrated during validations with various references (GABA, pentylenetetrazole…) • Excellent sensitivity (>85%) and predictivity (>85%) • Functional test • High through-put screening (manual / automate) • Limited compound needs (5 mg) • Cell transfected with rat/human clone, possibly use of iPS in the future • Cons/Limitations • Solubility • Many other receptors/channels implicated

9. SEIZURE/CONVULSION LIABILITYBrain slice electrophysiology Hippocampal slice • BRAIN SLICE ELECTROPHYSIOLOGY(hippocampus, cortex) is aimed at determinate drug-induced changes in local brain electrical activity (extracellular recording, focal EEG) using a MEA (microelectrode array) • Results: spontaneous spiking (rate, seizure-like discharges…) and evoked EPSP (area under the curve, slope, amplitude…) • Pros • High scientific reliability demonstrated with various references (GABA, pentylenetetrazole, psychostimulants…) • Excellent sensitivity (86%) and predictivity (100%) • Rapid functional test with mechanistic approaches • Limited compound needs (30 mg) • Various slices: cortex, hippocampus, possibly use of iPS layers in the future • Cons/Limitations • Solubility • Translation from focal to generalized seizure • Medium/Low through-put for screening CA1 DentateGyrus CA3 Microelectrode array AUC analysis Spiking Spike sorting

10. Control Control 2.5mM 5mM 10mM 15mM 25mM 2.5mM 5mM 10mM 15mM 25mM Total Distance Moved (mm)High Velocity 900 800 700 *** *** 600 *** 500 distance moved (mm) *** *** *** *** *** *** *** *** 400 *** *** 300 *** *** *** *** * *** 200 *** *** *** ** ** ** * * * 100 0 0-5 5-10 10-15 15-20 20-25 25-30 30-35 35-40 40-45 45-50 50-55 55-60 Time (min) SEIZURE/CONVULSION LIABILITY Zebrafish convulsion assay • ZEBRAFISH (ZF) CONVULSION ASSAYisaimedat quantitative detection of convulsion-likelocomotor patterns (velocity>20 mm/s) by videotracking of ZF larvae (Ethovision XT) and qualitative scoring of behavioralalterationsusingvideorecording • Results: safelevel and convulsion threshold(µM) • Pros • High scientificreliability for convulsantswith discrimination of psychostimulants • Good sensitivity (77%) and sufficientpredictivity (63%) • In vivo test (integrated model) • Medium throughput test / Limited compound needs (50mg, n=16 larvae/group) • Wide and staged range of concentrations frompharmacology to MTD (5-6 concentrations) • Cons/Limitations • Solubility • Interpretation as regards blood or brain concentrations ——— Control ——— PTZ, 2.5mM ——— PTZ, 5 mM ——— PTZ, 10 mM ——— PTZ, 15 mM ——— PTZ, 25 mM

11. SEIZURE/CONVULSION LIABILITY PTZ convulsion assay in rodents • PENTYLENETETRAZOLE (PTZ) CONVULSION ASSAYisaimedatmimicking a seizure-sensitive population (Ph1 safety of anyabnormal « healthyvolunteers  ») by quantitative detection of anyproconvulsanteffects in naiverodents (before PTZ) then in non-naiverodentsbrought to a sub-convulsant level (with a subconvulsant PTZ dose). • Results:safelevel and convulsion thresholdi.e. dose (mg/kg) and blood (eventuallybrain) level (ng/mL) withmyoclonus, clonic, clonico-tonic or tonic convulsions / per se and +PTZ effects) • Pros • High scientificreliability for convulsants • In vivo test (integrated model) • Medium throughput test / Quitelimited compound needs (100 mg, n=8/group, 3 doses) • IV route to exacerbateCmaxeffects or any Clinical route / rat or mouse / otherproconvulsant agents canbeused to test anyothermechanism (strychnine, pilocarpine…) • Kindlingprotocolsavailable • Cons/Limitations • PK interaction with PTZ

12. SEIZURE/CONVULSION liabilityEEG telemetry in rats • EEG TELEMETRYin freely-moving rats (implanted with supradural cortical electrodes) + video-recording to detect / discriminate convulsions for quantitative detection of number / frequency / delay of abnormal EEG morphologies (spike-wave trains, seizures and/or convulsion…) / pre-ictal changes • Results:safe level, seizure and convulsion threshold i.e. dose (mg/kg) and blood (eventuallybrain) level (ng/mL) with abnormal EEG morphologies • Pros • High scientific reliability for convulsants + discrimination of other CNS agents • Excellent sensitivity (100%) and predictivity (100%) • In vivo test (integrated model) = gold standard • IV route to exacerbateCmaxeffects or any Clinical route • Kindlingprotocolsavailable • Cons/Limitations • Low throughput test • Medium compound needs (300 mg, 3 dose-groups, n=6 rats/group) • Artefacts, ambiguous interpretation • Number of recording leads Spectral analysis with Theta Waves in EBs period EEG EBs

13. SEIZURE/CONVULSION LIABILITY Adapted FOB test in rodents • ADAPTED FOB ASSAYisaimedat quantitative and qualitative detection of any convulsant effects in naiverodentsthatmaybemissed in a routine FOB procedure due to limited duration of observation. Animals are observedindividually in an open-field for an extendedperiod of time after administration (1-3 hour) followed by additionalkinetic observations during the first 6-8h • Results:safe level and convulsion threshold i.e. dose (mg/kg) and blood (eventuallybrain) level (ng/mL) withnumber of animalsversus controlspresentingtremors, myocloni, clonic, clonico-tonic, tonic convulsion or mortality • Pros • Good scientificreliability • Improvedsensitivity vs classic FOB • IV route to exacerbateCmaxeffects or any Clinical route • Antidote caneasilybetested • Administration schedulescanbetested • Kindlingprotocols • Cons/Limitations • Medium compound needs (300-1000 mg, 3 dose-groups, n=8 rats/group)

14. SEIZURE/CONVULSION LIABILITY Videorecording assay in rats • VIDEORECORDINGisaimedat quantitative and qualitative detection of any convulsant effects in naive rats thatmay have been missed in othersstudies. Animals are individuallyvideorecorded in a Phenotyper cage 24h/day (IR camera, Ethovision XT, The Observer) with no limitation in duration. Followingdeathdiscovery, videocanberewound back to the animal death and causes, concommittant or precedingclinicalsignscanbevisualized and diagnosed • Results:number of death and preceding causes observed (diagnostic) • Pros • Good scientificreliability • Diagnostic tool • Kindlingprotocols • Cons/Limitations • Low throughput test

15. SEIZURE LIABILITY Strategy Preprog Program Program Candidate Pre Cd Preclinical Preclinical Lead Lead Lead Optimization Lead Optimization Candidate Selection Candidate Selection Development Development Identification Identification • Target assessment Predicting • Secondary Pharmacology • (extended panel) • Secondary Pharmacology • (short panel) Derisking Understanding • Derisking Off-Target pharmacology (IC50, ago/antago) • - GABA A • - Other targets • TOX acute preliminary rodent / non rodent • Preliminary PK Rodent (non rodent) • Brain slices rodent • Zebrafish convulsion test • PTZ convulsion test • TOX DRF rodent • TOX DRF rodent • Follow-up test (Brain slices, EEG, Videotracking, adapted FOB, antidote search) • EEG rodent

16. Evolving models • In silico • Lack of specific in silico modelspredictingeffects on CNS receptors / channelslinkedwithseizure or convulsion • Lack of integrated in silico modelspredictingdirectlyseizure or convulsion • Electrophysiologyusinghuman stem cells or iPS • patch clamp • microelectrodearray • High content analysis • Bioimaging • mainlyneurotoxicity • Biomarkers • resultsevolvingfromepilepsyresearch … • but mainly changes in biomarkers as a consequence of a seizure (not prediction)

17. Questions to the audience • Preclinical translation frompreclinical to clinical • Confidence in predictivity • Gaps • Improvements • Clinical evaluation • Confidence on availabletools • Gaps • Improvements