Workshop Models for proconvulsant drugs and precautions for entry into man Jean-Pierre MARTINOLLE
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Transcript of Workshop Models for proconvulsant drugs and precautions for entry into man Jean-Pierre MARTINOLLE
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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
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100
0.20
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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
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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
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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