Presentedby

Blessing Umoudit

Outline

• Background• In vitro electrophysiological approaches• In vitro CNS tissue models for electrophysiology• Emerging Models used in CNS safety pharmacology studies• Personal review• Conclusion

Image source: http://azneuromod.com/central-nervous-system-may-contribute-to-dpn/

Background• Central Nervous System • The ICH S7A guidelines recognized the CNS as a central pillar within safety

pharmacology • CNS drugs possess high attrition rates due to adverse effects• CNS adverse effects are mediated predominately by off-target drug activity• Altered neuronal function and network communication

• In response • There is a growing need to perform more comprehensive CNS safety

testing prior to first-in-human trials

Background cont’d Ion channels

Play a fundamental role in inter and intracellular communication and neuronal excitability

Subjected to rigorous studies

Some off target interactions are associated with ion channels HERG, Voltage-gated sodium channels (5CN5A),

GABA type a receptor

In vitro Electrophysiological Approaches

• Patch Clamp technique• The “Gold standard”

• Clamping is by forming a giga-ohm seal between plasma membrane and a glass or quartz micropipette

• Used to measure biophysical and pharmacological activity of ion channels on millisecond timescales.

MANUAL AUTOMATIC

VersatileLacks the versatility and quality of manual technique

Yields low throughput High throughput

High maintenance cost and level of expertise

Inexpensive

Manual vs Automated Patch Clamp Technique

• Impalement Techniques• Forcible penetration of the membrane of large cells with sharp

micropipettes

• Xenopus laevis oocytes are commonly used• Common e.g. is two electrodes voltage clamping (TEVC)• In TEVC, one electrode acts as a dedicated membrane potential

sensor and the other as a current injector.

• Smaller electrodes mean less disruption in membrane and cytosolic ionic composition compared to patch clamp techniques

In vitro Electrophysiological Approaches Cont’d

In vitro Electrophysiological Approaches Cont’d

Common in vitro electrophysiological methods.

Expression systems

and immortalized

cell linesDissociated neuronal primary

cultures

3D - Neuronal Models

Brain slice models

In Vitro CNS Tissue Models for Electrophysiology

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Expression Systems and Immortalized Cell Lines

• Are heterologous and recombinant expression systems(cells/cell lines) maintained in culture for an extended period of time.

• Non neuronal cells (e.g. Xenopus oocytes) were initially used

• Neural Stem Cells (NSC) are now being used• They possess Na+, K+ and Ca2 + currents in accordance with the known

patterns described for in vivo neuronal system

Dissociated Neuronal Primary Cultures

• Are neuronal cells mechanically and enzymatically dissociated from various brain regions

• Advantage• They retain most of their functionality in vitro• Success is dependent on specific requirement • This is dictated by the neurons cultured (e.g.

age of donor)

• Disadvantage• Cannot be maintained in culture for extended

periods and so would need to be freshly isolated regularly

Enzymatic dissociation of neuronal cells. (Hai-Yan Wu et.al 2014)

3D Neuronal Models• Consist of a co-mixture of different neuronal and non- neuronal sources• When grown in an in vitro 3D environment, neuronal cells demonstrate

better survivability as they are more similar to in vivo “3D” models.• Highly compatible with a number of electrophysiological techniques• Limitation• Cells within the centre of these models are exposed to less oxygen

and are often nutrient deprived

Brain Slice Models• Acute brain slice • Most accurate and the most common slice model used • Allows the use of intra and extracellular electrophysiological

techniques in a near in vivo situation• However, slice induced damage, lack of oxygen/glucose, bacterial

contamination limit the lifespan of these models

• Organotypic slice cultures• Capable of maintaining brain slice in culture on a stable substratum

over prolonged periods of time.• However, it does not retain its shape and thickness as it flattens to 3D

models

Animal Model Considerations• Rodents (mice and rats) are the primary species used in CNS

investigations• Transgenic mice enable the study of pharmacological drugs on

variety of conditions (e.g. Alzheimer’s disease, epilepsy etc.)• These models have shown good preclinical and safety testing

potential as they express the exact protein observed in the human situation

http://www.ddw-online.com/chemistry/p102797-zebrafish

https://en.wikipedia.org/wiki/African_clawed_frog

http://www.criver.com/products-services/basic-research/find-a-model/sprague-dawley-rat

Emerging Models in CNS safety pharmacology studies• Receptor Profile safety screening -Targets included on a recommended minimal panel for testing - Generates drug response profiles

• High-throughput techniques

• Liability Testing• Seizure liability assessment• Memory loss

Liability testing• Seizure LiabilityA number of pharmaceutical drugs targeting the CNS have been associated with seizures.

Case study : Minaprine an antidepressant withdrawn from market in 1996 due to an increase in clinical incidence of convulsions.

• Memory lossStudies have shown that compounds such as benzodiazepines that cause deficit in human memory have also led to dramatic changes in vitro.

Personal review• It is important to monitor these techniques to determine if

they are justifiable

• Suitability in preclinical studies (3RS)

• Predictive validity

Conclusion• Predictive power of in vitro models for CNS safety is low• The CNS is a complicated system and so there is need for

more enhanced means of studying it• Interspecies differences in ion-channel expression,

neuroanatomy and drug metabolism also reduce the predictive power in humans• Advancements have been made to techniques to make them

more applicable to in vivo conditions

Thank you!!!

Questions?