Whole bloody module Flashcards
1
Q
Benzodiazepines
A
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
2
Q
Barbiturates
A
Pentobarbital
Positive Allosteric Modulator of GABAaR, agonist at higher doses
Anxiolytic, anticonvulsant, insomnia, epilepsy
3
Q
Bicuculline
A
GABAaR competitive antagonist
Excitatory, mimics epilepsy
4
Q
a1-GABAaR
A
Mediates sedation, anti-convulsion and addiction
5
Q
a2-GABAaR
A
Anxiolytic, muscle relaxant, additive?
6
Q
a3-GABAaR
A
Muscle relaxant
7
Q
a5-GABAaR
A
Amnesia (Cognition), muscle relaxant
NAMs promote cognition
Expressed extrasynaptically in hippocampal CA1 neurons, media’s tonic current
8
Q
L655,708 (a5IA or R0493851)
A
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
9
Q
GABA phasic and tonic inhibition
A
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)
10
Q
H101R Mutation
A
Causes loss of benzodiazepine-sensitivity in a1,2,3,5-GABAaRs
11
Q
R101H Mutation
A
Causes gain of benzodiazepine-sensitivity in a4,6-GABAaRs
12
Q
Heat hyperalgesia
A
Paw withdrawal upon exposure to defined radiant heat,
Measure paw withdrawal
13
Q
Mechanical sensitisation
A
Mechanical stimulus used, Von Frey filaments of varying size used to spike foot. Measures paw withdrawal
14
Q
Cold allodynia
A
Time spent lifting, shaking or licking paw after drop of acetone on an injured paw
15
Q
a2-GABAaRs expressed densely in:
A
Lamina II/III
16
Q
Inflammatory pain model
A
Zymosan A
Injected into hind paw, assessed by measuring paw withdrawal
17
Q
Neuropathic (chronic) pain Model
A
Sciatic nerve tie off
18
Q
L838,417
A
GABAa selective PAM
Acts as partial agonist/PAM of a2,3,5-GABAaR
NAM/IA of a1-GABAaR
Analgesic, anxiolytic
No sedative effect
19
Q
How to assess affinity and efficacy of GABA drugs?
A
Transcription with particular GABAaR DNA, then either:
Measure binding affinity by homogenising and using radioligand binding assay
Measure efficacy by using whole-cell-clamp
20
Q
Tests for Anxiety
A
Elevated plus maze
Light/dark maze
Common component of many neurological disorders
21
Q
Tests for fear
A
Cued fear conditioning (amygdala)
Contextual fear conditioning (amygdala and hippocampus)
PTSD, phobias
22
Q
Tests for attention (human)
A
Wisconsin Card Sort
Behavioural flexibility test
Schizophrenia, ADHD
23
Q
Test for attention (mouse)
A
Attentional set-shifting
Dependant of prefrontal cortex
24
Q
Depression
A
Learned helplessness
Tail suspension test
Forced swim test
Dependant on anterior cingulate cortex
Depression, bipolar disorder, OCD
25
Locomotor Activity
Activity box
Parkinson’s disease, muscular atrophy
26
Locomotor coordination
Rotarod
Skilled reaching
Balance beam
Parkinson’s disease, Huntington’s disease
27
Sensory perception
Von Frey Test
Temperature sensitivity
Pain in Parkinson’s disease, chronic pain (neuropathic pain)
28
Learning
Morris water maze
Radial arm maze
Pair-associated learning
Autism, amnesia, Alzheimer’s disease
29
Memory
Spontaneous alteration
Novel object recognition
Autism, amnesia, Alzheimer’s disease
30
Procedural memory
How?
Striatum dependant
Not usually effected in most neurological disorders
31
Declarative memory
What?
Semantic (facts)
Episodic (events)
Usually affected by Parkinson’s disease, Alzheimer’s disease and dementia
Dependant on hippocampus
32
Translational
Trained to mimic human test
33
Naturalistic
Uses relevant or innate skill or preference of the animal
34
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
35
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
36
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
37
Pair Associated Learning (human)
Pairs shown, humans must recall other object after being shown one half
38
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
39
Pair associated learning (mouse, translational)
Mouse is trained to use touchscreen exactly the way humans do,
Require a lot of training
40
Phase I trials
Safety in healthy humans
| 1-2 years
41
Phase II trails
Efficacy of treatment
| 2-5 years
42
Phase III trials
Large scale study of efficacy and safety
3-10 years
43
Phase IV trials
Ongoing review of safety in the very long term in real patients
44
Drug discovery phase
High-throughput screening of compounds on target receptor
| Narrowing down and refinement of successful candidates
45
Preclinical development
Pharmacokinetic optimisations
46
What scale to use for plotting concentrations?
Logarithmic
47
How to shiver logarithmic concentrations?
Serial dilution
48
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
49
TR-FRET
Time resolved fluorescent energy transfer
50
Z’ factor
Tests robustness of assay
```
Z>0.6 = excellent
0.6>Z>0.3 = workable
Z<0.3 = not workable
```
51
Assays should be:
SRRM
Simple
Robust
Relevant
Miniaturisable
52
How to identify a ligand’s mechanism?
Concentration-response curve and compare it to natural ligand
53
Fraction bound =
Fraction bound = [D]/([D] + Kd)
54
Linear regression:
| Y =
Y = 1/(1+(Kd/x))
55
How to calculate geometric mean:
GM = ‘cube route’(a x b x c)
56
pKd =
pKd = -log(Kd)
57
siRNA
Can knockdown expression of genes
58
First order elimination
Is a straight line on log plot
59
Kel =
Kel = ln(2)/(t1/2)
60
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
61
Clearance
| Cl =
Cl = Kel , Vd
Therefore
Cl = (ln(2) . Vd) / (t1/2)
62
Free drug hypothesis
Pharmacological activity is dependant on free drug
Things affecting free drug conc are:
Membrane
Lipids
Protein
63
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
64
Transcellular
Flow through cell
| More hydrophobic compounds, active transport can contribute
65
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
66
The effect of CSF?
Quite negligible as blood:CSF interface is 5000x smaller than blood:brain
67
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
68
P-Glycoprotein (P-gp)
Endothelial ATPases which active pump molecule back out to prevent transcellular permission
Only on apical side of cell (blood)
69
PAMPA assay
Parallel artificial membrane permeability assay
Uses hexadecane membrane
Can be converted to use a cell monolayer
70
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
71
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
72
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.
73
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
74
Idiopathic epilepsy (primary)
Cannot be ascribed to particular cause or incident
| Eg benign neonatal convulsions or juvenile myoclonic epilepsy
75
Symptomatic epilepsy (secondary)
Associated with trauma, neoplasm (abnormal growth), infection, developmental abnormalities, cerebrovascular disease.
76
Generalised epilepsy
Eg grand mal or petit mal
77
Focal epilepsy
Eg partial seizures
78
EEG use
Electroencephalograph, records brain waves
79
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
80
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
81
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
82
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
83
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
84
Atonic seizure
Sudden loss of posture leading to collapse
85
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
86
Infantile Spasms
Syndrome of bilateral attacks of tied recurrent myoclonic jerks.
90% of patients have attacks before 1 year of age
87
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
88
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
89
Maximal shock test
Model for partial and tonic-clonic
Also, pilocarpine (mAChR antagonist) administered for 14 days
Ameliorated by phenytoin
90
Pentylenetetrazol-induced clonic seizure
GABAaR antagonist
| Model for generalised seizures, especially absence
91
Minimal metrazol-induced Seizures
Model myoclonic seizures
92
Electrical kindling
Model for complex partial seizures
| Repeated focal electrical stimulation
93
Mouse models
Star-gazer
Lethargic
Tottering
Models for generalised seizures (similar to PTZ)
94
Juvenile Myoclonic epilepsy
nAChR alpha7 subunit
95
Autosomal dominant nocturnal frontal lobe epilepsy
nAChR alpha 4, beta 2 subunit
96
Generalised epilepsy with Febrile seizures
GABAaR alpha 1, gamma 2, delta subunits
97
Generalised epilepsy with Febrile seizures with broader APs
Na+ channel 1A, 1B, 2A subunits
98
Benign familial neonatal convulsions
KCNQ2/3
99
Partial epilepsy/episodic atoxia
KCNA1
100
Only inhibitory neurons have:
GAD - GABA decarboxylase
101
Clonazepam
Benzodiazepine used against absence and some tonic and clonic/myoclonic seizures
102
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
103
Phenytoin
Block tetanus firing through increasing Na+ channel inactivation
Reduced neurotransmitter release
104
Carbamazepine
Block titanic firing through increased Na+ channel inactivation
Potentiates GABA response
Tricyclic antidepressant
105
Valproate
Block titanic firing through increased Na+ channel inactivation
Inhibit T-type Ca2+ channel
Inhibit GABA breakdown
Used against absence seizures
106
Ethosuxamide
Inhibits T-type Ca2+ channel
Inhibits Na+ v channels
Used to treat absence and myoclonic seizures
107
Lamotrigine
Na+ v channel blocker
Inhibits release of excitatory amino acids
Phenytoin, primidone, carbamazepine reduce t1/2, valproate increases t1/2
108
Topiramate
Blocks Na+ channels
Inhibits Kainate receptors
Enhances GABA action
Also anti-migraine
109
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
110
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
111
Levetriacetam
Binds to synaptic vesicle protein SVA2
Inhibits presynaptic Ca2+ channels
Reduces neurotransmitter release
Used to treat partial onset, myoclonic or tonic-clonic seizures
112
Acetazolamide
Carbonic anhydride inhibitor
Reduces local pH to decreased NMDAR activity
Used to treat menstrual related epilepsy and refractory epilepsy
