in vitro and in vivo electrophysiology for robust decision making in CNS drug discovery programs Robert E. Petroski, PhD MAY 28, 2020
WELCOME TO TODAY’S WEBINAR! Marketing & Communication Manager Neuroservices-Alliance … in CNS drug discovery programs 2 3 in vitro and in vivo electrophysiology Synaptic transmission ADHD behavioral for robust decision making and plasticity assays May 28 June 11 June 25
WELCOME TO TODAY’S WEBINAR • Ask your questions in the question box • Bob will answer your questions during the 10 min Q&A following his presentation
NEUROSERVICES-ALLIANCE in vitro data • Electrophysiology in vivo data • Electrophysiology • Behavior Translational data between species • Rodent • Non Human Primate • Human Worldwide Studies PhD scientists Years of cumulated clients experience
MEET TODAY’S PANELIST Scientific Liaison Neuroservices-Alliance
PATCH CLAMP REVOLUTIONIZED NEUROPHYSIOLOGY
OUTLINE Opportunities for CNS Drugs CNS Drug Discovery Workflow Electrophysiology Assays
THERAPEUTIC OPPORTUNITIES FOR CNS DRUGS V. Pain I. Psychiatric disorders I. Neuropathic pain i. Anxiety II. Visceral pain ii. PTSD iii. Depression iv. Schizophrenia IV. Epilepsy v. Bipolar I. Doose syndrome II. Dravet syndrome III. Lafora disease IV. Landau-Kleffner syndrome II. Neurodegeneration V. Lennox-Gastault syndrome VI. Others I. Alzheimer’s disease II. Parkinson’s disease III. Huntington’s disease III. Neurodevelopmental disorders IV. Motor neuron diseases I. Autism spectrum V. Others II. Angelman’s syndrome III. Down syndrome IV. Rett syndrome V. Others
THERAPEUTIC STRATEGIES Antisense Oligonucleotide Therapy U N IQU E E X P E R T IS E Small IN N E U R OP H A R MA C OL OGY molecules Gene Therapy Biologics Stem Cell Therapy
CNS DRUG DISCOVERY WORKFLOW Pre-clinical Pre-clinical Target Research Target Research IND IND Screening Screening Lead optimization Lead optimization development development Stage gates GO / NOGO
CNS DRUG DISCOVERY WORKFLOW Pre-clinical Target Research IND Screening Lead optimization development Stage gates GO / NOGO
CNS DRUG DISCOVERY WORKFLOW Pre-clinical IND Target Research Screening Lead optimization development I. Target validation II. Target signaling pathways III. Target mechanisms IV. Target biomarkers
R6/2 (Huntington’s) HFS TARGET RESEARCH 2.2 Normalized fEPSP MEA Hippocampal slice 1.8 amplitude CA1 1.4 CA3 1.0 WT R6/2 dentate gyrus 0 10 20 30 40 50 60 70 Time (min) Tg2576 (Alzheimer’s) HFS 2.2 field Excitatory Post-Synaptic Potentials (fEPSP) Normalized fEPSP 1.8 amplitude 1.4 WT 1.0 baseline fEPSPs Tg2576 after TBS 0 10 20 30 40 50 60 70 Time (min)
CNS DRUG DISCOVERY WORKFLOW Pre-clinical Target Research IND Screening Lead optimization development I. HTS assays Engineered “kit assays” using surrogate endpoints
CNS DRUG DISCOVERY WORKFLOW Pre-clinical Target Research IND Screening Lead optimization development I. Cellular assays II. Brain slices assays
SIGNALS / ENDPOINTS Field excitatory postsynaptic potentials (fEPSPs) Evoked excitatory postsynaptic currents (EPSCs) Evoked inhibitory postsynaptic currents (IPSCs) Miniature spontaneous excitatory postsynaptic currents (mEPSCs) Spontaneous action potential firing rate Miniature spontaneous inhibitory postsynaptic currents (mIPSCs) resting membrane potential (Vm) Input resistance (Rm) Long-term potentiation (LTP) Evoked action potential threshold (rheobase) Long-term depression (LTD) After hyperpolarizing potential (AHP) Chemical LTD INTRISIC EXCITABILITY SYNAPTIC TRANSMISSION SYNAPTIC OF NEURONS BETWEEN NEURONS PLASTICITY INTRISIC EXCITABILITY VOLTAGE-GATED ION LIGAND-GATED OF NETWORKS CHANNELS ION CHANNELS Population spikes (pop spikes) Glutamate receptors: AMPA Epileptiform discharges (EDs) Glutamate receptors: NMDA Na currents Oscillations GABA A receptors K currents Nicotinic receptors Ca currents
GABA A CURRENT FROM CULTURED NEURON 3 uM GABA (100 ms) puffer pipette recording electrode 250 pA 2 sec
INDIPLON POTENTIATES NATIVE GABA A RECEPTOR CURRENTS
MEA RECORDING OF SPONTANEOUS FIRING IN HIPPOCAMPAL SLICES Pirenzepine Pirenzepine (100 nM) (100 nM) + carbachol (µM) carbachol (µM) 2.0 1 0.1 0.3 0.5 1 3 1.5 (normalized) Firing rate 1.0 0.5 0.0 0 20 40 60 80 100 120 140 Time (min) 5 1 µM carbachol 2.0 Carbachol Carbachol + 4 100 nM Pirenzepine (normalized) 1.5 Firing rate ( normalised) Firing rate 3 1.0 2 0.5 EC 50 = 0.23 µM 1 EC 50 = 1.29 µM n H = 1.78 0 0.0 10 30 50 70 -8 -7 -6 -5 Time (min) log [Carbachol], M
PATCH CLAMP FROM PREFRONTAL CORTEX SLICES pyramidal neuron (layer V) 50 mV 500 ms PV positive interneuron (layer 2/3)
CNS DRUG DISCOVERY WORKFLOW Pre-clinical IND Screening Lead optimization Target Research development I. In vivo target engagement II. Target engagement in human slices
IN VIVO RECORDING METHODS
IN VIVO SIGNALS SINGLE UNITS (HIGHPASS FILTERED >300 HZ) LFPs ARE SLOW (LOWPASS FILTERED <200 Hz)
LOCAL FIELD POTENTIALS and QUANTITATIVE EEG LFPs are phasic or oscillatory • Phasic signals include evoked potentials, sharp waves, ictal spikes • Oscillatory signals include slow wave sleep, hippocampal theta, ripples LFPs are a great marker of large-scale synchronization • LFPs in rodents can be a preclinical biomarker of target engagement • LFPs in rodents are a translational biomarker for clinical EEG
PRE-ICTAL SPIKES IN HIPPOCAMPUS OF FREELY-MOVING Tg2576 MICE Pre-ictal spikes Pre-ictal spikes increased during REM inter-ictal spike rate (Hz) 0.08 0.06 0.04 0.02 0.00 wake SWS REM Levetiracetam reduces pre-ictal spikes REM SWS WAKE inter-ictal firing rate (Hz) 0.020 inter-ictal firing rate (Hz) 0.020 inter-ictal firing rate (Hz) 0.08 0.015 0.015 0.06 0.010 0.010 0.04 p= 0.026 0.005 0.005 0.02 p= 0.030 p= 0.069 0.000 0.000 0.00 VEH LEV VEH LEV VEH LEV
Spontaneous EPSCs recorded in voltage clamp HUMAN BRAIN SLICES Brain slices 20 pA Tissue from brain 50 ms resection Action potential train Recorded in current clamp Hippocampal pyramidal 20 mV neuron from human brain slice 500 ms
HUMAN IPSC-DERIVED NEURONS Pre-clinical Lead optimization IND Target Research Screening development i P S C - D E R I V E D N E U R O N S • Commercial iPSCs • Your proprietary iPSCs M E A S U R E M E N T S • Current clamp (excitability) • Voltage clamp (ion channels)
OUR VALUE PROPOSITION SHORT QUALITY EXPERTISE TURNAROUND > 500 CUMULATED YEARS ROBUST AND OF EXPERIENCE IN LARGE BANDWIDTH AND One-Stop-shop CRO REPRODUCIBLE DATA NEUROPHARMACOLOGY TOP LEVEL PLATFORMS For CNS INNOVATION INTERACTION FLEXIBILITY INTERACTIVE AND ACTIVE R&D STEP-BY-STEP EXPERT SUPPORT ALL PROGRAMS STUDY PLANS ALONG THE STUDY We co-design Every scientific question is unique custom solutions with our clients and so is every solution
ANY QUESTIONS?
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