Ionotropic receptors: therapeutics

Question 1

What is the pathophysiology of epilepsy?

Answer 1

Imbalance in Glu/GABA transmission → hyperexcitability

Synchronous bursts of APs (firing APs constantly causes loss of signal-noise ratio)

Status epilepticus can lead to excitotoxicity

iGluRs may play role in triggering seizure (triggering may involve loss of GABA activity?)

iGluRs involved in actual seizure activity (definitely involved in synchronising activity / passing AP bursts between neurons)

Question 2

What are the current mechanisms for licensed epilepsy treatments?

Answer 2

Potentiate GABA synapses - (diazepam)

Inhibit VGSCs (particularly blockers that have use-dependent effects i.e. block channels that are more active) - (phenytoin)

Inhibit VGCCs (ethosuximide)

Activate VGPC - (retigabine)

Inhibit NDMARs: FELBAMATE

Inhibit AMPA: PERAMPANEL

Question 3

How does felbamate work?

Answer 3

Not entirely understood but seems to be NAM of both GluN1 and GluN2 receptors

*also PAM of GABA-A receptors

Question 4

How does perampanel work?

Answer 4

AMPA NAM (binds between LBD and TMD)

• not selective for any subtype of AMPAR
• only licensed for PARTIAL seizures in UK
• side effects: somnolence, dizziness, fatigue, falls - if blocking AMPA across brain, will get widespread side effects e.g. cerebellum - decreasing balance

Question 5

What could provide a potential new approach for treating epilepsy? What evidence has been used to suggest this method?

Answer 5

Targeting AMPARs that are associated with Ɣ-8

Forebrain regions important in epilepsy (cortex / hippocampus / temporal lobe)

mRNA expression used to indicate TARP expression in mouse/rat (although less accurate as may not be a linear relationship between them):
Ɣ-2 very widespread (avoid)
Ɣ-3; forebrain but also other areas
Ɣ-4; not really expressed in cortex/Hi
Ɣ-8; more selective in cortex & hippocampus, less in other areas

Question 6

What are TARPs involved in?

Answer 6

Traffic AMPARs to surface (e.g. KO Ɣ-2 - lose AMPARs in cerebellar cells)

Ɣ-2, Ɣ-3, Ɣ-4, Ɣ-8 interact with AMPA receptors
(the other 4 interact with calcium channels)

Change glutamate affinity, desensitisation, deactivation & pharmacology - effectiveness of some drugs is changed by TARP expression

Question 7

What is a FLIPR assay?

Answer 7

Fluorescence Imaging Plate Reader

Common cellular assay in drug development (screening)

Grow cells (HEK / Chinese hamster ova etc.) which express protein of interest in plate with 384 wells - calcium sensitive fluorescent dye loaded into cells - add as solution then wash off excess

Machine can add many drugs + measures fluorescence -can get responses from all wells in 6 mins

First give standard conc of each drug to screen compounds, then refine the compound (add groups / substitutes etc.)

Question 8

How has FLIPR been used to find drugs selective for Ɣ-8-associated AMPARs?

(Kato et al)

Answer 8

Expressed GluA1(o) in cells with TARPs, and used GYKI (NAM) as control (inhibits response in all cells)

Screened 1000s of compounds for differentiation between Ɣ-2 & Ɣ-8 expressing cells

LY3130481 (antagonist) = conc-dependent inhibition of AMPA response: greater inhibition with Ɣ-8 than Ɣ-2. Also went on to show selective inhibition (Ɣ-8) in flip + flop GluA1/2/3/4

BUT, cultured cells, not relevant to real neurons

Question 9

After testing with FLIPR (cultured cells), what was LY3130481 tested on?

Answer 9

Rat hippocampal slice: add glutamate every 30s, then add increasing concentrations of drug - AMPA response inhibited - get IC50

Still potent (although slightly less potent than in cultured cell lines)

Question 10

How was it demonstrated that LY3130481 is dependent on Ɣ-8 in real neurons?

Answer 10

Mouse hippocampal slice: Ɣ-8 KO

No inhibition of AMPA response in KO

In KO: still get other AMPARs going to the surface - but other TARPs

Question 11

How was the potential role of LY3130481 in epilepsy tested? What did this show?

Answer 11

Inducing seizures in animals: pre-treat with drug then measure % that had convulsions

Vehicle: 100% had seizures

LY3130481 acted as anticonvulsant in animal model
*More potent than GYKI or perampanel

Question 12

Why is LY3130481 better than GYKI / Perampanel?

Answer 12

• Given orally as crosses BB-barrier (GYKI and perampanel = I.P. injections, may not cross as well)
• More potent (GYKI/perampanel needs higher concentrations to stop seizures)
• No motor impairment - actually shows non-significant increase in motor function (perampanel impairs motor function at doses with anti-epileptic activity as inhibits AMPARs across the brain)

Question 13

What is the exact mechanism of LY3130481?

Answer 13

Directly binds to Ɣ-8 TARP, even in the absence of AMPAR subunits (shown by crystal structures)

Not yet known how it changes AMPAR function..

Question 14

Outline pathophysiology of stroke

Answer 14

Majority: ischaemic (artery thrombosis) Remainder: haemorrhagic (cerebral artery rupture) - CT scan needed to diagnose

Three key processes in ischaemia:

1) Inflammation (cells die/burst → release inflammatory mediators)
2) Free radical production
3) Rise in extracellular glutamate concentration (causes excitotoxicity - persistent neuronal activation and NMDARs activated)

Question 15

How is ischaemic stroke currently treated?

Answer 15

Tissue plasminogen activator (tPA) within 3 hours

• Only for ischaemic, link with functional benefits in patients
• If >3 hours, restoring flow can spread free radicals to other areas causing more damage

Question 16

What are the key elements in the cascade leading to cell death in stroke (necrosis vs apoptosis)

Answer 16

1) Na⁺ + Ca²⁺ influx upsets osmotic balance → NECROSIS → cell lysis & release of contents including glutamate & inflammatory mediators

Calcium influx also causes..

2) Mitochondrial impairment → less ATP → free radical production (oxidative stress) → CYTOCHROME C release (APOPTOSIS initiator)
3) Glutamate release from presynaptic terminals

Finally..

4) REVERSAL of glutamate TRANSPORTERS causes even more glutamate release!
(due to loss of function of Na⁺/K⁺ exchanger which usually maintains physiological Na⁺/K⁺ concentration

Question 17

What steps result in Ca overload in stroke?

Answer 17

Glu activates AMPARs - removes Mg²⁺ block of NMDARs: rise in [Ca²⁺]i

This depolarisation activates VDCCs, further increasing [Ca²⁺]i

Activation of mGlu1 or 5 also increases [Ca²⁺]i

Internal Ca²⁺ stores e.g. endoplasmic reticulum and the Ca²⁺ efflux pump cannot cope with rise in [Ca²⁺]i
*resting calcium inside cell usually ~100nM tightly controlled by efflux pump and pump into stores like endoplasmic reticulum

Question 18

How does Ca overload cause cell death in stroke?

Answer 18

Mitochondria take up Ca²⁺, this causes uncoupling of ATP production (less ATP) and formation of reactive oxygen species (ROS - can lead to cell death)

Ca²⁺ dependent enzymes activated indiscriminately leading to cell death:

• lipase;
• protease;
• endonuclease; break down DNA
• phospholipase A2
• nitric oxide synthase

Question 19

How do free radicals contribute to stroke pathophysiology?

Answer 19

Mitochondria produce Superoxide (O₂⁻):
highly reactive + produces other free radicals → neuronal death

• SOD (destroys superoxide) cannot cope with excessive production during ischaemia
• nNOS is activated by Ca²⁺ and produces free radical nitric oxide (NO*)
• Inducible NOS (iNOS) activated in response to inflammation: produces much more NO* than nNOS

NO* can react with superoxide (O₂⁻) to form peroxynitrite which damages DNA and proteins, is membrane permeable so spreads damage to neighbouring neurons

Question 20

In stroke, what causes an increase extracellular glutamate? And what is the effect of this?

Answer 20

Activation of PLA₂ by Ca²⁺ causes ARACHIDONIC ACID production, which inhibits glutamate transporter and produces free radicals

*Activation of NMDARs, AMPARs and KARs increases [Na⁺]i, this leads to NECROSIS

Calcium also disrupts mitochondria: low [ATP] causes Na⁺/K⁺ exchanger failure→ disruption of Na⁺/K⁺ ion balance causes reversal of EAAT (starts to pump out Glu)

Question 21

What is the anti-excitotoxic hypothesis? (stroke)

Answer 21

Protect neurons by immediate treatment with a GluR antagonist or Glu release inhibitor

Question 22

What possible anti-excitotoxic drugs for stroke have been looked at?

Answer 22

NMDAR antagonists:
- shown to reduce extent of brain lesions (neuroprotective) in animal models of ischaemic stroke (e.g. tie off cerebral/carotid artery- give drugs, then relieve tension so that blood flow returns)

Competitive NMDAR antagonists, channel blockers, glycine site antagonists & NBQX (AMPAR antagonist) all failed in clinical trials

Question 23

What effects of anti-excitotoxic drugs resulted in ending the clinical trials?

Answer 23

Lack of neuroprotective effect (therapeutic brain concentrations may not have been achieved e.g. BBB crossing issues)

Adverse effects such as hallucinations, effects on memory and ataxia (loss of motor coordination)

Question 24

Why have NMDAR antagonists failed in clinical trials?

Answer 24

1) Should give within 2 hours, but treatment windows were extended to several hours
2) 6-24 hours post stroke, minor, sustained [Glu] increase may promote neuronal survival by NMDAR-DEPENDENT transcriptional activation of PRO-SURVIVAL GENES
3) Loss of WM (axons) + glia: large impact on outcome of ischaemia in humans? GluN2C / GluN2D may play a role in WM injury + 1st generation competitive antagonists were GluN2A/GluN2B selective
4) GluN3 NMDARs are expressed on oligodendrocytes- current competitive NMDAR antagonists may not block these effectively (as some don’t bind glutamate)
5) As NMDARs not the only proteins involved in neuronal cell death -may need combination of different drugs

Question 25

Why are NMDAR antagonists that are selective for certain subunits now a focus for stroke treatment?

Answer 25

GluN2(A-D) subunits have different distributions in CNS, so GluN2 selective antagonists may have fewer side effects

*GluN2B or GluN2D-containing NMDARs implicated in producing excitotoxicity, whereas GluN2A-containing NMDARs contribute to neuroprotective signalling in animal models of TBI and ischaemia

Question 26

What are examples of SELECTIVE NMDAR antagonists that are being considered for stroke treatment?

Answer 26

1) GluN2-selective allosteric modulators (e.g. NAMs like ifenprodil)
2) Dual acting compounds that are GluN2A PAMs and GluN2D NAMs
* may have fewer side effects as not completely blocking, less hallucination/psychoses

Question 27

What is changing in stroke/brain injury treatment trials?

Answer 27

New methods are in place to allow treatment of acute brain injury in clinical trials within the 2 hour therapeutic time window

Multimodal approaches to stroke treatment e.g. use of glutamate receptor antagonists, as well as anti-inflammatory drugs and free radical scavengers

Question 28

What is prion disease

Answer 28

Transmissble spongiform encephalopathies (TSEs) - neurogenerative diseases

Misfolded forms of cellular prion protein (PrPC) are crucial in their genesis and progression

Question 29

What did Khosravani et al (2008) show? (prion disease)

What can be concluded from this?

Answer 29

Transient NMDA exposure (in vivo) causes increased lesion size and excitotoxic cell death in the hippocampus of PrP KO mice

*PrPc KO mice have enhanced + prolonged NMDAR currents due to upregulation of GluN2D - these effects can be reversed by over expression of exogenous PrPc.

Enhanced activity of GluN2D NMDARs leads to excitotoxicity (both in vitro + in vivo models) - therefore - PrPc is neuroprotective by INHIBITING GluN2D subunit expression (misfolded PrPC is not protective)

Therefore, could give GluN2D antagonists as neuroprotectants?

Question 30

What did Hizue et al (2005) show?

neuropathic pain

Answer 30

Partial sciatic nerve ligation (PSL) to induce mechanical allodynia (chronic pain model)

GluN2D knockout mice have a paw withdrawal threshold similar to sham wild type mice (sciatic nerve not ligated)

This resistance to mechanical allodynia was not observed with GluN2A KOs

Therefore, GluN2D NMDARs involved in neuropathic pain transmission and selective antagonists may be potential treatments?

Question 31

What is the evidence for a role of NMDA receptors in depression?

Answer 31

Skolnik et al (2009): positive effect of ketamine (0.5mg/kg) in depressed patients within 3 hours + sustained for at least 3 days

BUT: is this via NMDAR? ketamine also binds to other things

GluN2B selective antagonists - antidepressant effects for as long as 30 days in patients unresponsive to SSRIs

Question 32

Further Reading: examples of NMDA antagonists that failed clinical trials?

Answer 32

*MK801 did not reach human clinical trials (safety concerns - rat histopatholgical changes). Also thought to be neuroprotective due to hypothermia. Buchan et al. found it was no longer neuroprotective when temp controlled.

SELFOTENEL - increase in early mortality (but only 13% were treated within 3 hours). no morbidity benefit

APTIGANAL hydrochloride - Phase IIB - same problems as above.

Question 33

Further reading: what is another approach for stroke treatment that is not an NMDAR antagonist or NAM

Answer 33

Drugs that change the interaction between NMDA and intracellular signalling mechanisms such as PSD-95.

Transient global ischaemia shows to change the interaction between NMDAR and PSD-95 protein.

Also clinical trials for Magnesium Sulfate starting in USA