Whole bloody module Flashcards
Benzodiazepines
Diazepam, Flunitrazepam
Positive allosteric modulators of GABAa Receptor
Bonds to BZ site, on alpha-gamma interface, dependant on H101 residue
Does not bind to alpha-4,6 isoforms as residue it R101
Anxiolytic, insomnia, epilepsy
Barbiturates
Pentobarbital
Positive Allosteric Modulator of GABAaR, agonist at higher doses
Anxiolytic, anticonvulsant, insomnia, epilepsy
Bicuculline
GABAaR competitive antagonist
Excitatory, mimics epilepsy
a1-GABAaR
Mediates sedation, anti-convulsion and addiction
a2-GABAaR
Anxiolytic, muscle relaxant, additive?
a3-GABAaR
Muscle relaxant
a5-GABAaR
Amnesia (Cognition), muscle relaxant
NAMs promote cognition
Expressed extrasynaptically in hippocampal CA1 neurons, media’s tonic current
L655,708 (a5IA or R0493851)
Binds to a1,2,3,5 GABAaRs
NAM/IA of a5-GABAaR, no affect on a1,2,3
Benzodiazepine antagonist of a1,2,3
Cognitive enhancer
GABA phasic and tonic inhibition
Phasic Mediated by synaptic GABAaRs by triggered neurotransmitter release
(Seen as individual spikes or hyperpolarisation)
Tonic mediated by extrasynaptic GABAaRs, and also GABAbRs to some extent, residual GABA in ECF mediates this
(Seen as a constant hyperpolarisation)
H101R Mutation
Causes loss of benzodiazepine-sensitivity in a1,2,3,5-GABAaRs
R101H Mutation
Causes gain of benzodiazepine-sensitivity in a4,6-GABAaRs
Heat hyperalgesia
Paw withdrawal upon exposure to defined radiant heat,
Measure paw withdrawal
Mechanical sensitisation
Mechanical stimulus used, Von Frey filaments of varying size used to spike foot. Measures paw withdrawal
Cold allodynia
Time spent lifting, shaking or licking paw after drop of acetone on an injured paw
a2-GABAaRs expressed densely in:
Lamina II/III
Inflammatory pain model
Zymosan A
Injected into hind paw, assessed by measuring paw withdrawal
Neuropathic (chronic) pain Model
Sciatic nerve tie off
L838,417
GABAa selective PAM
Acts as partial agonist/PAM of a2,3,5-GABAaR
NAM/IA of a1-GABAaR
Analgesic, anxiolytic
No sedative effect
How to assess affinity and efficacy of GABA drugs?
Transcription with particular GABAaR DNA, then either:
Measure binding affinity by homogenising and using radioligand binding assay
Measure efficacy by using whole-cell-clamp
Tests for Anxiety
Elevated plus maze
Light/dark maze
Common component of many neurological disorders
Tests for fear
Cued fear conditioning (amygdala)
Contextual fear conditioning (amygdala and hippocampus)
PTSD, phobias
Tests for attention (human)
Wisconsin Card Sort
Behavioural flexibility test
Schizophrenia, ADHD
Test for attention (mouse)
Attentional set-shifting
Dependant of prefrontal cortex
Depression
Learned helplessness
Tail suspension test
Forced swim test
Dependant on anterior cingulate cortex
Depression, bipolar disorder, OCD
Locomotor Activity
Activity box
Parkinson’s disease, muscular atrophy
Locomotor coordination
Rotarod
Skilled reaching
Balance beam
Parkinson’s disease, Huntington’s disease
Sensory perception
Von Frey Test
Temperature sensitivity
Pain in Parkinson’s disease, chronic pain (neuropathic pain)
Learning
Morris water maze
Radial arm maze
Pair-associated learning
Autism, amnesia, Alzheimer’s disease
Memory
Spontaneous alteration
Novel object recognition
Autism, amnesia, Alzheimer’s disease
Procedural memory
How?
Striatum dependant
Not usually effected in most neurological disorders
Declarative memory
What?
Semantic (facts)
Episodic (events)
Usually affected by Parkinson’s disease, Alzheimer’s disease and dementia
Dependant on hippocampus
Translational
Trained to mimic human test
Naturalistic
Uses relevant or innate skill or preference of the animal
Radial arm maze
Reference memory - which arms are baited
Working memory - which arms have already been visited in the current trial
Require food and animal scent controls
Barnes Maze
Holes around edge with one leading to home box
Controls for odour and scent required, as well as proximal cues (objects) for animal to reference
Morris Watermaze
Opaque water with one platform
Naturalistic and rats swim to escape cold water
No scent controls required
Proximal cues required
Human analogue is being shown a position on a computer screen, then entering a 3D arena eg tent, and placing object on the same spot
Pair Associated Learning (human)
Pairs shown, humans must recall other object after being shown one half
Pair associates learning (mouse, naturalistic)
Choice trial
Mouse searches arena with hole in it and eventually finds ‘food x’, mouse repeated this and finds ‘food y’ at different location
Mouse can then be cues by food x or food y to go to the specific burial spot
Pair associated learning (mouse, translational)
Mouse is trained to use touchscreen exactly the way humans do,
Require a lot of training
Phase I trials
Safety in healthy humans
1-2 years
Phase II trails
Efficacy of treatment
2-5 years
Phase III trials
Large scale study of efficacy and safety
3-10 years
Phase IV trials
Ongoing review of safety in the very long term in real patients
Drug discovery phase
High-throughput screening of compounds on target receptor
Narrowing down and refinement of successful candidates
Preclinical development
Pharmacokinetic optimisations
What scale to use for plotting concentrations?
Logarithmic
How to shiver logarithmic concentrations?
Serial dilution
High-Throughput Screening
Thousands to millions of chemicals in library
Screening of ligands hypotheses to have an effect by computational studies
TR-FRET used with europium labelled antibody
~1% hit rate
TR-FRET
Time resolved fluorescent energy transfer
Z’ factor
Tests robustness of assay
Z>0.6 = excellent 0.6>Z>0.3 = workable Z<0.3 = not workable
Assays should be:
SRRM
Simple
Robust
Relevant
Miniaturisable
How to identify a ligand’s mechanism?
Concentration-response curve and compare it to natural ligand
Fraction bound =
Fraction bound = [D]/([D] + Kd)
Linear regression:
Y =
Y = 1/(1+(Kd/x))
How to calculate geometric mean:
GM = ‘cube route’(a x b x c)
pKd =
pKd = -log(Kd)
siRNA
Can knockdown expression of genes
First order elimination
Is a straight line on log plot
Kel =
Kel = ln(2)/(t1/2)
Volume of distribution
Vd =
Represents what volume the drug would have to be distributed in to give the observed plasma concentration
Vd (L) = dose (mg) / concentration (mg/L)
Usually related to body weight!
Vd (L/kg) = dose (mg/kg) / concentration (mg/L)
0.05L/kg = blood volume
0.3kg/L = extracellular water volume
0.5-2L/kg = broad distribution
>2L = accumulation in tissue
Clearance
Cl =
Cl = Kel , Vd
Therefore
Cl = (ln(2) . Vd) / (t1/2)
Free drug hypothesis
Pharmacological activity is dependant on free drug
Things affecting free drug conc are:
Membrane
Lipids
Protein
Partition coefficient
Kp =
Kp u.u =
Kp = brain conc / plasma conc
If over 1 then higher concentration in brai
Kp u.u = unbound brain conc / unbound blood conc
Better measure of brain penetration as it takes into account brain has 20x more lipid and half the amount of protein, so free drug can be affected greatly
Transcellular
Flow through cell
More hydrophobic compounds, active transport can contribute
Paracellular
Flows through gap between cells,
Usually small molecular molecule/ions, hydrophilic compounds
In BBB this gap is very tight due to tight junctions formed by caludins and occludins, so it is inhibited
The effect of CSF?
Quite negligible as blood:CSF interface is 5000x smaller than blood:brain
Blood Brain Barrier
Important to protect ionic concentrations in CNS to maintain neuronal function.
Complex structure of endothelial, astrocyte and pericyte cells
Allows distribution of very small molecules such as dissolved gasses
Has active uptake mechanisms for glucose and amino acids
P-Glycoprotein (P-gp)
Endothelial ATPases which active pump molecule back out to prevent transcellular permission
Only on apical side of cell (blood)
PAMPA assay
Parallel artificial membrane permeability assay
Uses hexadecane membrane
Can be converted to use a cell monolayer
Measuring P-gp activity
MDCK cells expressing P-gp monolayer used to separate two fluids, Concert measured on each side to calculate an efflux ratio
Passive diffusion should have ratio of ~1, if P-gp is pumping it out (higher conc in apical fluid), then ratio will be >1
Measuring CNS penetration in humans
Conc in CSF approx equal to conc in brain ISF, as there are minimal barriers, it can give a good indication
Only accurate if active transport does not take place
Sampling is invasive
Positron Emission
Proton from unstable parent nucleus degrades into a neutron (beta decay), emitting a positron and neutrino.
Electron and positron collide and annihilate one another, this emits two antiparallel photons (gamma waves)
PET detector reads photos emitted, signal requires co-incident if both photons - it allows for mapping of location
Isotopes that decay by beta positive decay include: C11, N13, O15, F18
Short lived, therefore radioisotopes have to be prepared on site.
Epilepsy definition
Group of CNS disorders in which recurrent seizures occurs due to chronic underlying processes, affecting motor, sensory and autonomic outputs
Seizures are paroxysmal events, due to abnormal synchronous discharges from a population of CNS neurons
Idiopathic epilepsy (primary)
Cannot be ascribed to particular cause or incident
Eg benign neonatal convulsions or juvenile myoclonic epilepsy
Symptomatic epilepsy (secondary)
Associated with trauma, neoplasm (abnormal growth), infection, developmental abnormalities, cerebrovascular disease.
Generalised epilepsy
Eg grand mal or petit mal
Focal epilepsy
Eg partial seizures
EEG use
Electroencephalograph, records brain waves
Simple partial seizure
No impairment of consciousness, can be confined to single muscle group/limb.
Brain locus is identifiable
KCNA1 mutation
Treated with:
Gabapentin - Ca v channel alpha2-delta, increases GABA biosynthesis by up GAD and up BCAT
Vigabatrin - inhibits GABA transaminase, half life of biological activity longer than elimination half life and levels of GABA-T take up to 6 days to recover
Complex partial seizures
Consciousness impaired, confusion and stumbling, with automatisms resembling ticks
Treated with:
Gabapentin - Ca v channel alpha2-delta, increases GABA biosynthesis by up GAD and up BCAT
Vigabatrin - inhibits GABA transaminase , half life of biological activity longer than elimination half life and levels of GABA-T take up to 6 days to recover
Partial seizures secondary generalised
Partial seizure followed immediately my generalised tonic-clonic seizure
Treated with:
Gabapentin - Ca v channel alpha2-delta, increases GABA biosynthesis by up GAD and up BCAT
Vigabatrin - inhibits GABA transaminase, half life of biological activity longer than elimination half life and levels of GABA-T take up to 6 days to recover
Generalised tonic-clonic seizures
Aka grand mal
Tonic rigidity followed by tremor
Clinic phase relaxations are longer causing muscle jerking
Treated by:
Levetiracetam - binds to synaptic vesicles glycoprotein SV2A and inhibits presynaptic Ca2+ channels reducing neurotransmitter release
Absence seizure
Aka petit mal
10-45s, up to 100 a day.
Altered consciousness, mild clonic spasms may occur.
Automatisms May confuse diagnosis with complex partial.
Start in childhood and patients often suffer mental retardation.
Treated by:
Ethosuxamide - inhibit T type Ca2+ channels in thalamic neurons, inhibit Na+ v channels
Valproate - increase Na+ channel inactivation, inhibit T type Ca2+ channels, inhibit GABA breakdown
Atonic seizure
Sudden loss of posture leading to collapse
Myoclonic seizure
Isolated clonic jerks associated with multiple spikes in EEG, rhythmic series of clonic seizures
Treated by:
Ethosuxamide - inhibit T type Ca2+ channels in thalamic neurons, inhibit Na+ v channels
Levetiracetam - binds to synaptic vesicles glycoprotein SV2A and inhibits presynaptic Ca2+ channels reducing neurotransmitter release
Infantile Spasms
Syndrome of bilateral attacks of tied recurrent myoclonic jerks.
90% of patients have attacks before 1 year of age
Status Epilipticus
Continuous or repetitive seizure over 30 mins in durations
Inadequate treatment results in brain damage or death
Can occur by non-compliance with treatment program, or by medicated patients suffering fever/infection which can lower serum drug conc
Can be induced by sudden withdrawal of benzodiazepines or barbiturates
Epileptic recruitment of surrounding neurons
Raised K+ conc in ECF causing depolarisation
Raised Ca2+ in presyanptic terminal causing neurotransmitter release
Depolarisation-induced NMDAR activation increasing intracellular Ca2+ conc
Maximal shock test
Model for partial and tonic-clonic
Also, pilocarpine (mAChR antagonist) administered for 14 days
Ameliorated by phenytoin
Pentylenetetrazol-induced clonic seizure
GABAaR antagonist
Model for generalised seizures, especially absence
Minimal metrazol-induced Seizures
Model myoclonic seizures
Electrical kindling
Model for complex partial seizures
Repeated focal electrical stimulation
Mouse models
Star-gazer
Lethargic
Tottering
Models for generalised seizures (similar to PTZ)
Juvenile Myoclonic epilepsy
nAChR alpha7 subunit
Autosomal dominant nocturnal frontal lobe epilepsy
nAChR alpha 4, beta 2 subunit
Generalised epilepsy with Febrile seizures
GABAaR alpha 1, gamma 2, delta subunits
Generalised epilepsy with Febrile seizures with broader APs
Na+ channel 1A, 1B, 2A subunits
Benign familial neonatal convulsions
KCNQ2/3
Partial epilepsy/episodic atoxia
KCNA1
Only inhibitory neurons have:
GAD - GABA decarboxylase
Clonazepam
Benzodiazepine used against absence and some tonic and clonic/myoclonic seizures
Additional mechanism for epilepsy to occur and be treated:
BBB leakage allows albumin to enter brain ISF, bind to astrocyte TGFb, results in downregulation of inward-rectifying K+ channels, causing hyperexcitability
Decreased expression of glutamate transporters EAAT-1,2 occurs in brain after seizure
Seizure can induce inflammation by cytokines production and brain-derived neurotrophic factor (BDNF) can sustain hyperactivity
E-prostanoid-2 receptor antagonists can help prevent neuronal injury after status epilipticus
Astrocytic adenosine kinase degrades adenosine, which is a neuron derives-anticonvulsant, this can be inhibited
mTOR inhibition with rapamycin
Seizure induced expression of P-glycoprotein can limit effectiveness of drug therapy
Phenytoin
Block tetanus firing through increasing Na+ channel inactivation
Reduced neurotransmitter release
Carbamazepine
Block titanic firing through increased Na+ channel inactivation
Potentiates GABA response
Tricyclic antidepressant
Valproate
Block titanic firing through increased Na+ channel inactivation
Inhibit T-type Ca2+ channel
Inhibit GABA breakdown
Used against absence seizures
Ethosuxamide
Inhibits T-type Ca2+ channel
Inhibits Na+ v channels
Used to treat absence and myoclonic seizures
Lamotrigine
Na+ v channel blocker
Inhibits release of excitatory amino acids
Phenytoin, primidone, carbamazepine reduce t1/2, valproate increases t1/2
Topiramate
Blocks Na+ channels
Inhibits Kainate receptors
Enhances GABA action
Also anti-migraine
Gabapentin
Ca v channel alpha2-delta,
Increases GABA biosynthesis by up GAD and up BCAT
Little protein bound or metabolised (interacts with cimetidine, Mg and Al antacids
Used to treat partial seizures with or without secondary generalisations
Also use in chronic pain management
Vigabatrin
inhibits GABA transaminase
half life of biological activity longer than elimination half life and levels of GABA-T take up to 6 days to recover
Reduces plasma conc of primidone
Used to treat partial seizures
Levetriacetam
Binds to synaptic vesicle protein SVA2
Inhibits presynaptic Ca2+ channels
Reduces neurotransmitter release
Used to treat partial onset, myoclonic or tonic-clonic seizures
Acetazolamide
Carbonic anhydride inhibitor
Reduces local pH to decreased NMDAR activity
Used to treat menstrual related epilepsy and refractory epilepsy
