A brief overview of a research article on CNS safety

1. Presented
by
Blessing Umoudit

2. 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/

3. 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

4. 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

5. 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
Versatile
Lacks 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

6. • 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

7. In vitro Electrophysiological Approaches Cont’d
Common in vitro electrophysiological methods.

8. Expression
systems
and
immortalized cell lines
Dissociated neuronal primary
cultures
3D - Neuronal Models
Brain slice models
In Vitro CNS Tissue Models for
Electrophysiology
Increasingcomplexity

9. 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

10. 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)

11. 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

12. 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

13. 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

14. 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

15. Liability testing
• Seizure Liability
A 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 loss
Studies have shown that compounds such as benzodiazepines
that cause deficit in human memory have also led to dramatic changes
in vitro.

16. Personal review
• It is important to monitor these techniques to determine if
they are justifiable
• Suitability in preclinical studies (3RS)
• Predictive validity

17. 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

18. Thank you!!!
Questions?