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Workshop

Models for proconvulsant drugsand precautions for entry into man

Jean-Pierre MARTINOLLESafety Pharmacology Advisory Group

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SEIZURE / CONVULSIONFacts …

Phase Preclinical Phase I Phase I-III Phase III/ Marketing

Post-Marketing Post-Marketing

Information: Causes of attrition Causes of attrition Serious ADRs Causes of attrition Causes of attrition ADRs on label Serious ADRs Withdrawal from sale

Source: ABPI (2008) Car (2006) Sibille et al. (1998) ABPI (2008) Olson et al. (2000) BioPrint® (2006) Budnitz et al. (2006) Stevens & Baker (2008)

Sample size: 156 CDs stopped 88 CDs stopped 1,015 subjects 63 CDs stopped 82 CDs stopped 1,138 drugs 21,298 patients 47 drugs

Cardiovascular 24% 27% 9% 35% 21% 36% 15% 45%

Hepatotoxicity 15% 8% 7% 29% 21% 13% 0% 32%

Haematology/BM 3% 7% 2% 3% 4% 16% 10% 9%

Nervous system 12% 14% 28% 2% 21% 67% 39% 2%

Adapted from Redfern WS et al. SOT 2010 Poster 1081

● 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

DrugCite 2013

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SEIZURE / CONVULSIONDefinition / Risk

● 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

Chemicalproperties

SEIZURERISK

Kindling

Co-medication

Healthy

Cmax

Pharmacologicalclass

Compoundrelated

Known

Indication

SeizureSensitivity

DiseaseHistory

History

AcuteSeizure

CNS activity

On Target

Off Target

Patient

Populationrelated

Unknown

PreviousSeizure

Undetected

Metabolite

ChronicSeizure

Directconvulsant

Co-diseaseAdministration

related

Route

Single dose

Repeated dose

Structuralclass

BBB penetration

Facilitator

PgpMRP, BCRP

Brain concentration

● DRUG-INDUCED SEIZURE● 1% (world) with epilepsy, 3-10% (world ) will experience seizures in their life

10% 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

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Preclinical Predictivity

● Seizure is undetectable without sophisticated methods like EEG or bio-imaging

● Convulsion is tricky to detect if only based on observation● Rare event● Rarely occuring in all species tested● Often resolving quickly thus not seen (if limited time of observation)● Not always leading to death thus no alertThen chance of detection generally increases with the muliplication of studies and species

but can lead to a late discovery of the risk !

RISK TYPE POTENTIAL MECHANISMS PRECLINICAL PREDICTIVITY

Following single administration BBB crossing, Primary/secondary pharmacology Following single and repeated administration, with no aggravation

BBB crossing, Primary/secondary pharmacology …

Following single and repeated administration, with aggravation

BBB crossing/BBB altered, Primary/secondary pharmacologyPotential neurotoxicityKindling / Brain adaptative changesBrain accumulation / ADME effects

Following repeated administration but not after single administration

Observed in specific population (age, gender, pathology)Observed with co-medication

Observed with treatment withdrawal

BBB crossing/BBB altered, Primary/secondary pharmacology Co-factorsADME effectsBrain adaptative changes

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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

Classical Preclinical package

Possible detection Limitations/ Comments

CNS Gross behavior (FOB) rodent

mainly clear convulsion not seizure, improved detection when IV route

Only males-rodent-young adultsSingle admin. / Limited observations

CNS Locomotor activity rodent

did not detect convulsion nor seizure except if during administration

Only males-rodent-young adultsSingle admin. / Very limited observations

Detect CNS properties possibly in relation to seizure (stimulants, sedative …)

CARDIO TelemetryRESPI assay, rodent/non rodent

did not detect convulsion nor seizure except if during administration

Only young adultsSingle admin. / Very limited observations

Possibly video in Telemetry non rodent to explain death

General TOX (acute, DRF, REG)rodent / non rodent

mainly clear convulsion not seizure, improved detection when IV route after single/repeated administration + brain/histology

Only young adultsVery limited observations / Rare video to explain unknown lethalityUnadapted histopathology (generally no brain structural changes visible)

Genotox packageReprotox package

did not detect convulsion nor seizure except if during administration

Not designed for

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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, any withdrawal

● 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))

● Brain penetration

● Secondary pharmacology

Drug class Black list

Alcohols, glycols

Anesthetics, local

Anesthetics, general

Antibiotics

Anticonvulsants

Anticholinergics

Antidepressants, cyclic

Antidepressants, other

Antifungals

Antihistaminics

Antineoplasics

Antiparasitics

AntiviralsAsphyxiants

Cardiovascular

Drugs of abuse/withdrawal

Hypoglycemics

Immunosuppressives

Muscle relaxants

Mushrooms derivatives

Neuroleptics

Neuromuscular blockers

NSAIDs

Opioids

Radiography contrastant

Sedative reversal agents

SympathomimeticsVaccines…

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SEIZURE / CONVULSIONSecondary pharmacology black list

Receptors Adrenergics α1A antago Adrenergics α2A antago Adrenergics α1B ago Adrenergics 1 ago Adrenergics 2 ago Adenosine A1 antago Adenosine A2 antago AMPA ago Benzodiazepine BZD antago Cannabinoids CB1 antago Channels Na (+) Channels K (-) Channels Ca (+) (L, T, N, P/Q) CRF1 ago Dopamine D1 ago Dopamine D2 antago Endothelin ETA ago GABA A antago GABA B ago GABA B antago Galanin GalR1/2 antagonist Glutamate mGlu1/5 ago Glutamate mGlu2/3/4/7/8 antago Glycine (stryc sens) antago Glycine (stryc insens) ago

Histamine H1 Histamine H2 antago Histamine H3 ago Kainate ago Muscarinic M1 ago NCX1 reverse mode (+) NCX3 reverse mode (+) Nicotinic ago Nicotinic ago NMDA ago NPY2/5 antagonist Oxytocin antagonist Opioid µ ago Opioid K antago Opioid ago Opioid NOP ago P2X7 ago Serotonine 5-HT1A antago Serotonine 5-HT1B ago Serotonine 5-HT2C ago Serotonine 5-HT3 antago Serotonine 5-HT7 ago Sigma 1 ago Vasopressin V1a agonist

Other Acetylcholinesterase (-) Adenosine kinase (-) PDE3/4/7 inhibitors Cytokine, Growth factors (+/-) Glutamate synthetase (-) Glutamate deshydrogenase (+) Melatonin MT1/2 ago/antago NO synthase inhibitor Phosphate activated glutaminase (+) Transporters GLU Transporters Glutamine Transporters GABA Transporters DA Transporters 5-HT Transporters NE

strong evidence

evidence

unclear evidence

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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

20

40

60

80

100

0.01 0.1 1 10Pentylenetetrazole (mM)

Rel

ativ

e C

urr

ent

(%)

100

0.20

0.40

0.60

0.80

1.00

0.1 1 10GABA (µM)

I/IM

ax

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SEIZURE/CONVULSION LIABILITYBrain slice electrophysiology

● 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

DentateGyrus

CA3

CA1

Hippocampal slice

Microelectrode array

AUC analysis

Spike sorting

Spiking

Stages:HTLPreCanMechanistic

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Total Distance Moved (mm)High Velocity

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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)

dis

tan

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ove

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mm

)

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● ZEBRAFISH (ZF) CONVULSION ASSAY is aimed at quantitative detection of convulsion-like locomotor patterns (velocity>20 mm/s) by videotracking of ZF larvae (Ethovision XT) and qualitative scoring of behavioral alterations using video recording● Results: safe level and convulsion threshold (µM)

● Pros ● High scientific reliability for convulsants with discrimination of psychostimulants

● Good sensitivity (77%) and sufficient predictivity (63%)

● In vivo test (integrated model)

● Medium throughput test / Limited compound needs (50mg, n=16 larvae/group)

● Wide and staged range of concentrations from pharmacology to MTD (5-6 concentrations)

● Cons/Limitations● Solubility

● Interpretation as regards blood or brain concentrations

SEIZURE/CONVULSION LIABILITY Zebrafish convulsion assay

——— Control

——— PTZ, 2.5mM

——— PTZ, 5 mM

——— PTZ, 10 mM

——— PTZ, 15 mM

——— PTZ, 25 mM

Control 2.5mM Control

15mM10mM5mM2.5mM 25mM15mM10mM5mM 25mM

Stages:HTLLTCPreCanMechanistic

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● PENTYLENETETRAZOLE (PTZ) CONVULSION ASSAY is aimed at mimicking a seizure-sensitive population (Ph1 safety of any abnormal « healthy volunteers  ») by quantitative detection of any proconvulsant effects in naive rodents (before PTZ) then in non-naive rodents brought to a sub-convulsant level (with a subconvulsant PTZ dose).

● Results: safe level and convulsion threshold i.e. dose (mg/kg) and blood (eventually brain) level (ng/mL) with myoclonus, clonic, clonico-tonic or tonic convulsions / per se and +PTZ effects)

● Pros● High scientific reliability for convulsants

● In vivo test (integrated model)

● Medium throughput test / Quite limited compound needs (100 mg, n=8/group, 3 doses)

● IV route to exacerbate Cmax effects or any Clinical route / rat or mouse / other proconvulsant agents can be used to test any other mechanism (strychnine, pilocarpine…)

● Kindling protocols available

● Cons/Limitations● PK interaction with PTZ

SEIZURE/CONVULSION LIABILITY PTZ convulsion assay in rodents

Stages:PreCanCandidatePreclinical

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SEIZURE/CONVULSION liabilityEEG telemetry in rats

● EEG TELEMETRY in 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 (eventually brain) 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 exacerbate Cmax effects or any Clinical route● Kindling protocols available

● Cons/Limitations● Low throughput test● Medium compound needs (300 mg, 3 dose-groups, n=6 rats/group)● Artefacts, ambiguous interpretation● Number of recording leads 0

50

100

150

200

250

300

350

10 20 30 40

PTZ (mg/kg IP), n= 6 rats

Nu

mb

er

of

Sp

ike T

rain

s (

1st

ho

ur

po

st

do

sin

g)

Spectral analysis with Theta Waves in EBs period

EEGEBs

Stages:PreCanCandidatePreclinical

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● ADAPTED FOB ASSAY is aimed at quantitative and qualitative detection of any convulsant effects in naive rodents that may be missed in a routine FOB procedure due to limited duration of observation. Animals are observed individually in an open-field for an extended period of time after administration (1-3 hour) followed by additional kinetic observations during the first 6-8h● Results: safe level and convulsion threshold i.e. dose (mg/kg) and blood (eventually

brain) level (ng/mL) with number of animals versus controls presenting tremors, myocloni, clonic, clonico-tonic, tonic convulsion or mortality

● Pros● Good scientific reliability

● Improved sensitivity vs classic FOB

● IV route to exacerbate Cmax effects or any Clinical route

● Antidote can easily be tested

● Administration schedules can be tested

● Kindling protocols

● Cons/Limitations● Medium compound needs (300-1000 mg, 3 dose-groups, n=8 rats/group)

SEIZURE/CONVULSION LIABILITY Adapted FOB test in rodents

Stages:PreCanCandidatePreclinical

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● VIDEORECORDING is aimed at quantitative and qualitative detection of any convulsant effects in naive rats that may have been missed in others studies. Animals are individually videorecorded in a Phenotyper cage 24h/day (IR camera, Ethovision XT, The Observer) with no limitation in duration. Following death discovery, video can be rewound back to the animal death and causes, concommittant or preceding clinical signs can be visualized and diagnosed● Results: number of death and preceding causes observed (diagnostic)

● Pros● Good scientific reliability

● Diagnostic tool

● Kindling protocols

● Cons/Limitations● Low throughput test

SEIZURE/CONVULSION LIABILITY Videorecording assay in rats

Optime code SNX

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SEIZURE LIABILITY Strategy

Lead Identification Lead Optimization Candidate Selection

Preclinical Development

CandidateLead

Identification Lead Optimization Candidate SelectionPreclinical

Development

Pre Cd

• PTZ convulsion test• TOX DRF rodent• TOX DRF rodent

• Target assessment

• Secondary Pharmacology (short panel)

• Secondary Pharmacology (extended panel)

• Derisking Off-Target pharmacology (IC50, ago/antago) - GABA A - Other targets

• Preliminary PK Rodent (non rodent)• Brain slices rodent• Zebrafish convulsion test

Preprog Program

•TOX acute preliminary rodent / non rodent

Program

• Follow-up test (Brain slices, EEG, Videotracking, adapted FOB, antidote search)

• EEG rodent

Predicting

Derisking

Understanding

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Evolving models

● In silico● Lack of specific in silico models predicting effects on

CNS receptors / channels linked with seizure or convulsion● Lack of integrated in silico models predicting directly

seizure or convulsion

● Electrophysiology using human stem cells or iPS● patch clamp● microelectrode array

● High content analysis● Bioimaging

● mainly neurotoxicity

● Biomarkers● results evolving from epilepsy research …● but mainly changes in biomarkers as a consequence of a seizure (not prediction)

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Questions to the audience

● Preclinical translation from preclinical to clinical● Confidence in predictivity● Gaps● Improvements

● Clinical evaluation● Confidence on available tools● Gaps● Improvements