Experiment #1
The Effects of Anticonvulsant
Agents on Mice
Group 1 and 2
Pharmacology B
December 12, 2006
Objective
• To determine certain anticonvulsant
agents can counteract the effects of strong
convulsants such as nicotine or can act as
prophylaxis against these agents.
• This will be done by injecting the mice with
combinations of both convulsant and
anticonvulsant drugs at varying intervals
Define Seizures:
• A seizure is a paroxysmal event due to
abnormal, excessive, hyper-synchronous
discharges from an aggregate of central
nervous system neurons.
Depending on the distribution of discharges, this
abnormal CNS activity can have various
manifestation, ranging from dramatic convulsive
activity to experiential phenomena not readily
discernible by an observer.
Mechanism of action
and anticonvulsant
effect of the three drugs
Phenobarbital
• Anticonvulsant effect: causes a "depression" of
the body's systems, mainly the central and
peripheral nervous systems and by virtue of this
they produce a wide spectrum of effects, from
mild sedation to anesthesia
• Phenobarbital is indicated in the treatment of all
types of seizures except absence seizures.
• It is the first line choice for the treatment of
neonatal seizures.
Phenobarbital: Mechanism of action
Class: Barbiturates
• Exact mechanism is unknown
• Enhancement of inhibitory process and
diminishing of excitatory transmission
Prolong the opening of Cl- channels
↓
Enhances GABA receptor mediated current
↓
Inhibit generation of action potential
Phenobarbital (Barbituates)
ADVERSE EFFECTS:
• Sedation and hypnosis (principal side effects)
• CNs effects: dizziness, nystagmus and ataxia
• In old aged patients, they cause excitement and
confusion
• In children, they cause paradoxical hyperreactivity
• Anesthsia
• Respiratory and cardiovascular depression
• Muscle relaxation
Phenobarbital Drug Interactions
•
Barbiturates
– Induce hepatic microsomal drug metabolizing enzymes
– Additive central nervous system depression with other central nervous
system depressants
Highly Predicable
Predictable
Not established
Increase metabolism:
Increase metabolism:
Increase metabolism:
•Central nervous system
depressants (additive)
•Beta adrenoceptor
blockers
•Calcium channel blockers
•Corticosteroids
•Delavirdine
•Doxycycline
•Estrogens
•Phenothiazine
•Quinidine
•Cyclosporine
•Methadone
•Protease inhibitors
•Sirolimus
•Tacrolimus
•Theophylline
Decrease metabolism of
Phenobarbital:
•Valproic acid
Phenytoin (Dilantin)
• Phenytoin reduces the maximal activity of
brain stem centers responsible for the
tonic phase of tonic-clonic (grand mal)
seizures.
• For the control of generalized tonic-clonic
and psychomotor (grand mal and temporal
lobe) seizures
• Prevention and treatment of seizures
occurring during or following neurosurgery.
Phenytoin (Dilantin)
ADVERSE EFFECTS:
• Nystagmus & loss of smooth extraocular pursuit movements (not indication
to decrease the dose)
• Diplopia & ataxia (indication to adjust the dose)
• Sedation at high doses
• Gingival hyperplasia
• Hirsutism
• Long term effects:
– Coarsening facial features & mild peripheral neuropathy (manifested by
diminished deep tendon reflexes in lower extremities
– Abnormalities of Vitamin D metabolism  osteomalacia
– Decrease in folate levels  megaloblastic anemia
•
Rare effects:
–
–
–
–
–
–
Skin rash due to hypersensitivity of the drug
Fever
Skin lesions
Lymphadenopathy
Causal relationship to Hodgkin’s disease
Hematologic complications (agranulocytosis)
Phenytoin: Mechanism of Action
• The primary site of action appears to be the
motor cortex where spread of seizure activity is
inhibited.
• Possibly by promoting sodium efflux from
neurons, phenytoin tends to stabilize the
threshold against hyper-excitability caused by
excessive stimulation or environmental changes
capable of reducing membrane sodium gradient.
• This includes the reduction of post-tetanic
potentiation at synapses. Loss of post-tetanic
potentiation prevents cortical seizure foci from
detonating adjacent cortical areas.
Dilantin Drug Interactions
– Induces hepatic microsomal drug metabolism
– Susceptible to inhibition of metabolism by CYP2C9
and to a lesser extent CYP2C19
Predictable
Drugs whose Decrease levels:
metabolism •Corticosteroids
is stimulated •Doxycycline
by dilantin
•Methadone
•Quinidine
Not established
Decrease levels:
•Mexiletine
•Theophylline
•Verapimil (Ca
channel blocker)
•Cyclosporine
•Estrogens
Dilantin Drug Interactions cont…
Predictable
Drugs that
inhibit dilantin
metabolism
Drugs that
enhance
dilantin
metabolism
Not Predictable
Not Established
Increase serum
phenytoin:
Increase serum
phenytoin:
Increase serum
phenytoin:
•Amiodarone
•Chloramphenicol
•Elbamate
•Miconazole
•Disulfiram
•Isoniazide – problem
primarily with slow
acetylators of isoniazide
•Ticlopidine
•Capecitabine
•Fluorouracil
•Fluvoxamine
•Cimetidine
Decrease serum
phenytoin:
•Rifampin
Diazepam
• Diazepam is a frequently prescribed medication to treat
anxiety and stress.
• In emergency care, it is used to treat alcohol withdrawal
and grand mal seizure activity.
• It may also be used in conscious patients during
cardioversion and TCP to induce amnesia and sedation.
• Though the drug is still widely used as an anticonvulsant
because of its fast action, it is actually a relatively weak
anticonvulsant because of its short duration.
• Rapid IV administration may be followed by respiratory
depression and excessive sedation.
Diazepam: Mechanism of Action
Class: Benzodiazepines
•
Diazepam potentiates the effects of inhibitory
neurotransmitters (GABA), hyperpolarizing the
membrane potential and raising the seizure threshold
in the motor cortex.
1.
2.
Antagonism of serotonin
Increased release of and/or facilitation of gammaaminobutyric acid (GABA) activity
Diminished release or turnover of acetylcholine in the
CNS
3.
↓
Inhibit generation of action potential
Diazepam = Valium
ADVERSE EFFECTS:
• Somnolence
• Suppression of REM sleep or dreaming
• Impaired motor function, coordination, balance
• Dizziness
• Depression
• Anterograde amnesia (especially pronounced in higher
doses)
• Reflex tachycardia
• Rare paradoxical side effects can include: nervousness,
irritability, insomnia, muscle cramps, and in extreme
cases, rage, and violence.
Diazepam Drug Interactions
• Does NOT increase or decrease hepatic enzyme activity
• Does NOT alter the metabolism of other compounds
• Increases the central depressive effects of alcohol, other
hypnotics/sedatives (e.g. barbiturates), narcotics, and
other muscle relaxants
• Euphoriant effects of opioids may be increased, leading
to increased risk of psychological dependence
• Cimetidine, omeprazole, ketoconazole, itraconazole,
disulfiram, fluvoxamine, isoniazid, erythromycin,
probenecid, propranolol, imipramine, ciprofloxacin,
fluoxetine and valproic acid prolong the action of
diazepam by inhibiting its elimination.
• Oral contraceptives ("the pill") significantly decrease the
elimination of desmethyldiazepam, a major metabolite of
diazepam
Diazepam Drug Interactions cont…
• Rifampin, phenytoin, carbamazepine and phenobarbital
increase the metabolism of diazepam, thus decreasing
drug levels and effects
• Nefazodone can cause increased blood levels of
benzodiazepines
• Cisapride may enhance the absorption, and therefore
the sedative activity, of diazepam
• Small doses of theophylline may inhibit the action of
diazepam.
• Diazepam may block the action of levodopa (used in the
treatment of Parkinson's Disease)
• May alter digoxin serum concentrations
• May have interactions with diazepam include:
Antipsychotics (e.g. chlorpromazine) MAO inhibitors,
ranitidine
• Smoking tobacco can enhance the elimination of
diazepam and decrease its action
Procedure
All mice were injected intraperitoneally
with the specific drug in the table below.
Mouse
Initially
After 15 minutes
A
0.2cc of 3% Nicotine
Nothing
B
1mg Phenobarbital
Sodium
0.2cc of 3% Nicotine
0.2cc of 3% Nicotine
C
.3mg/kg Diazepam
D
5mg Dilantin
0.2cc of 3% Nicotine
E
0.2cc of 3% Nicotine
Phenobarbital - During
convulsions
F
0.2cc of 3% Nicotine
.3mg/kg Diazepam - During
convulsions
G
0.2cc of 3% Nicotine
1g Dilatin - During
convulsions
Parameters
Parameter
How where they used?
Hair On the Back
When the mouse felt the initial action of the
drug, the mouse’s hair on the back become
erected and the hairs where standing
sparsely from one another
Tail
Also few minutes followed by the hair, the
tail become very erected and later become
weaker
Movement/Mobility
The mouse started either by running fast in
circles (hyperactive) or by tremendously
slowing down mobility into a comatose state
or died.(hypoactive)
Whiskers
Similar to the tail, the initial action of the
drug also trigged the whiskers to appear as
erected.
Tapping of the feet
Part of the reaction to the drug was the
mouse was tapping the feet either really fast
or slowing down
Mouse A
= 15.5 g
Inject 0.2cc of 3% Nicotine intraperitoneally
Mouse A
= 15.5 g
Inject 0.2cc of 3% Nicotine intraperitoneally
Mouse B
Mouse B
= 15.5 g
Inject 30 mg/kg BW diazepam intraperitoneally
After 15 min, inject nicotine as above (0.2 cc of
3% intraperiotneally)
Mouse C
= 15.5 g
.3mg/kg Diazepam intraperitoneally
After 15 min, inject nicotine (0.2 cc of 3%
intraperiotneally)
TIME
OBSERVATION
0
0.05 cc diazepam injected (dose 0.05cc / 20 gm)
15 min
0.2 cc nicotine injected
16 min 26 sec
Evidence of seizure by hair standing & whiskers
straight, minimal movement but still on all fours
17 min 20 sec
Prior observations, straight tail, no foot twitching
27 min 35 sec
Prior observations & foot twitching
34 min
Mouse is more active exhibited by standing &
walking
35 min
Mouse lies down but still showing prior evidence of
seizures
36 min
Mouse rolled over but still alive
Mouse C
= 15.5 g
.3mg/kg Diazepam intraperitoneally
After 15 min, inject nicotine (0.2 cc of 3%
intraperiotneally)
= 16.7 g
Inject 5mg Dilantin intraperitoneally
After 15 min, inject nicotine (0.2 cc of
3% intraperiotneally)
Mouse D
TIME
(min)
OBSERVATIONS
TIME
(min)
OBSERVATIONS
0
Dilatin injected
25
Feet are just hanging but is able to turn
3
Slowing down, mild drowsy
27
Few feet movement
5
???
28
Started to bite and scratch face
6
Little bit of convulsion – few hairs
standing
29
On its belly with few head movement
30
Muscle twitching on back legs
9
Rapidly bitting tail
31
Rapid breathing but no movement
12
Moving in circle, tail bitting
32
Front leg twitching
13
Rapidly moving around but stopping
40
Still breathing
16
0.2 cc nicotine injected, more active
44
Still breathing, leg twitching
17
Rapidly bitting tail
45
Back leg moving
19
Convulsing
46
Body twitching
20
Lost its balance
50
Head is moving up
21
Lie on its side, unable to move one leg
23
Stop moving around but still tried to
move
Mouse D
= 16.7 g
Inject 5mg Dilantin intraperitoneally
After 15 min, inject nicotine (0.2 cc of
3% intraperiotneally)
Mouse E
.3mg/kg Diazepam intraperitoneally
After 15 min, inject nicotine (0.2 cc of 3%
intraperiotneally)
• 1:06 seconds, first reaction. After the next
med, 7 minutes was unresponsive
Mouse F:
= 16.7 g
Inject 0.2cc of 3% Nicotine
Then inject 3mg/kg Diazepam
during convulsions
• 0:10 seconds 1st reaction. After 6 minutes
significant decrease of behavior. After 9
minutes, unresponsive
Results
Mouse G
Weight= 16.8 g
Injected nicotine
Then dilatin 1g
.2cc of 3 %
Min
Actions
0 min
Injected nicotine
1 min 20 sec
Went into convulsion
1 min 21 sec
Injected dilatin
2 min 55 sec
Heavy and rapid breathing rate lying down no mvt
5 min
Heavy and rapid breathing rate no movement
12 min
Normal deep breathing rate no mvt
15min 34 sec
Normal breathing rate lying on its side
17 min 12 sec
Slow breathing rate no mvt
22 min 45 sec
Stopped breathing completely--- dead
Results
Mouse G
Weight= 16.8 g
Injected nicotine
Then dilatin 1g
.2cc of 3 %
Analysis – Mouse C
• Observations between the initial injection of Diazepam &
the Nicotine injection at the 15 min mark were not
recorded.
• It appeared that Diazepam, which was initially
administered to the mouse, was ineffective as an antiseizure drug when the mouse went into convulsion after
being injected with Nicotine at the 15 min mark.
• This could be indicative of the Diazepam wearing off
prior to the Nicotine being administered or the Nicotine
had a stronger effects which overwhelmed the effects of
Diazepam
• It appears that anti-seizure drugs are ineffective as a
preventive drugs to nicotine
#4 Discussion of Results of Experiment
Mouse
Expected Results
Actual Results
A
Convulsions
Convulsions
Sedation  Seizure
Sedation
Sedation
Seizure  Recovery  Seizure
Sedation  normal activity
Death
Nicotine 
phenobarbital
Convulsion  normal activity
Death
F
Convulsion  normal activity
Death
Convulsion  normal activity
Death
nicotine
B
Phenobarbitol 
nicotine
C
Diazepam  nicotine
D
Dilantin  nicotine
E
Nicotine  diazepam
G
Nicotine  dilantin
#4 Mechanism of Action of Nicotine
• At low to moderate doses,
nicotine is a cholinergic
agonist – it acts by
stimulating nicotinic
acetylcholine receptors.
• Nicotine can be absorbed through most of the
body's membranes. After nicotine is absorbed it
is distributed by the blood to a number of sites of
pharmacological action. The effects of nicotine
can be observed rapidly.
Can dianepam, phenobarbital and dilantin act as
a prophylaxis against nicotine? Why or why not?
GABA
• GABA acts at inhibitory synapses in the brain.
• GABA acts by binding to specific receptors in the plasma
membrane of both pre- and postsynaptic neurons
• This binding causes the opening of ion channels to allow
either the flow of negatively-charged chloride ions into
the cell or positively-charged potassium ions out of the
cell.
• This will typically result in a negative change in the
transmembrane potential, usually causing
hyperpolarization
Diazepam
• Mechanism of Action:
enhances the actions of GABA by causing GABA to bind more tightly
to the GABAA receptor. Increase frequency of Cl- channel opening.
※GABA GABAA – CNS
GABAB – Skeletal muscle
• It is believed that diazepam enhances the actions of GABA by
causing GABA to bind more tightly to the GABAA receptor
Phenobarbital
• Increases the action of the inhibitory
neurotransmitter, GABA in the brain. Also
appears to inhibit the release of glutamate
(an excitatory neurotransmitter) from nerve
endings. Increase duration of Cl- channel
opening
Dilantin
• Produces a voltage and frequency
dependent blockade of sodium channels in
rapidly discharging nerve cells. Thus, it
stops sustained repetitive firing such as
that occurring during a seizure. Because
of this it prevents the spread of seizure
discharge.
Nicotine
• Nicotine doesn't just stimulate the brain's "reward" centre, it also
shuts down the system that limits how long those rewards last.
• The brain's reward centres normally reinforce behaviours that are
good for you, such as eating when you're hungry.
• Nicotine hijacks the reward system by attaching to receptors on
nerve cells and triggering the release of dopamine, a
neurotransmitter which causes pleasant feelings.
• Nicotine also attaches to another receptor that triggers the release
of a chemical called GABA, which stops dopamine.
• The receptors keep releasing GABA until they run out and they can't
produce more for up to an hour after being exposed to nicotine.
• Without GABA, the body can't stop the pleasure signal caused by
nicotine.
#6 Different Types of Epilepsy and
management
Partial
(Focal)
Simple
Complex
Phenytoin
Phenytoin
Carbamazepine
Carbamazepine
Phenobarbital
Primidone
Primidone
#6 Different Types of Epilepsy and
management
Generalized
Tonic-clonic
(grand-mal)
Absence
(petit)
Mycoclonic
Febrile Seizures
in Children
Status
Epilepticus
Phenytoin
Valproic Acid
Valproic Acid
Diazepam
Phenytoin
Carbamazepine
Ethosuximide
Clonazepam
Phenobarbital
Clonazepam
Primidone
Valproi Acid
Phenobarbital
Diazepam
Lorazepam
Give the conclusion of the
experiment
Nicotine poisoning
• The LD50 of nicotine is 50 mg/kg for rats
and 3 mg/kg for mice. 40–60 mg can be a
lethal dosage for adult human beings. This
makes it an extremely deadly poison. It is
more toxic than many other alkaloids such
as cocaine, which has a lethal dose of
1000 mg.
Symptoms
•
•
•
•
•
•
•
•
vomiting and nausea, diarrhea
headaches
difficulty breathing
palpitations
stomach pains/cramps
seizures
weakness
increased drooling
Diagnosing
• Increased nicotine or cotinine (the nicotine
metabolite) is detected in urine or blood, or
increased serum nicotine levels occur.
• Historically, most cases of nicotine
poisoning have been the result of its use
as an insecticide; however, such use is
less frequent now than previously. Every
year many children go to the emergency
room after eating cigarettes or cigarette
butts. Sixty milligrams of nicotine has the
potential to kill an adult.
• which is about the amount of nicotine in
three or four cigarettes or half a cigar, if all
nicotine were absorbed. However, this
figure is higher in regular smokers,
although not drastically so Consuming
only one cigarette's worth of nicotine is
enough to make a toddler severely ill. In
some cases children have become
poisoned by topical medicinal creams
which contain nicotine.