Experiment #1The 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 actionand 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 actionClass: 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 hyper-

reactivity• 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:

•Central nervous system depressants (additive)

Increase metabolism:

•Beta adrenoceptor blockers•Calcium channel blockers•Corticosteroids•Delavirdine•Doxycycline•Estrogens•Phenothiazine•Quinidine

Decrease metabolism of Phenobarbital:•Valproic acid

Increase metabolism:

•Cyclosporine•Methadone•Protease inhibitors•Sirolimus•Tacrolimus•Theophylline

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 Not established

Drugs whose metabolism is stimulated by dilantin

Decrease levels:•Corticosteroids•Doxycycline•Methadone•Quinidine

Decrease levels:•Mexiletine•Theophylline•Verapimil (Ca channel blocker)•Cyclosporine•Estrogens

Dilantin Drug Interactions cont…

Predictable Not Predictable Not Established

Drugs that inhibit dilantin metabolism

Increase serum phenytoin:

•Amiodarone•Chloramphenicol•Elbamate•Miconazole•Disulfiram

Increase serum phenytoin:

•Isoniazide – problem primarily with slow acetylators of isoniazide•Ticlopidine

Increase serum phenytoin:

•Capecitabine•Fluorouracil•Fluvoxamine•Cimetidine

Drugs that enhance dilantin metabolism

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 ActionClass: Benzodiazepines

• Diazepam potentiates the effects of inhibitory neurotransmitters (GABA), hyperpolarizing the membrane potential and raising the seizure threshold in the motor cortex.

1. Antagonism of serotonin2. Increased release of and/or facilitation of gamma-

aminobutyric acid (GABA) activity3. Diminished release or turnover of acetylcholine in the

CNS

↓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

ProcedureAll mice were injected intraperitoneallywith the specific drug in the table below.

Mouse Initially After 15 minutes

A 0.2cc of 3% Nicotine Nothing

B1mg Phenobarbital

Sodium0.2cc of 3% Nicotine

C .3mg/kg Diazepam0.2cc of 3% Nicotine

D 5mg Dilantin 0.2cc of 3% Nicotine

E 0.2cc of 3% NicotinePhenobarbital - During

convulsions

F 0.2cc of 3% Nicotine.3mg/kg Diazepam - During

convulsions

G 0.2cc of 3% Nicotine1g 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

TailAlso few minutes followed by the hair, the tail become very erected and later become

weaker

Movement/MobilityThe mouse started either by running fast in

circles (hyperactive) or by tremendously slowing down mobility into a comatose state

or died.(hypoactive)

WhiskersSimilar to the tail, the initial action of the drug

also trigged the whiskers to appear as erected.

Tapping of the feetPart of the reaction to the drug was the

mouse was tapping the feet either really fast or slowing down

Mouse A = 15.5 gInject 0.2cc of 3% Nicotine intraperitoneally

Mouse A = 15.5 gInject 0.2cc of 3% Nicotine intraperitoneally

Mouse B

Mouse B= 15.5 gInject 30 mg/kg BW diazepam intraperitoneallyAfter 15 min, inject nicotine as above (0.2 cc of 3% intraperiotneally)

Mouse C= 15.5 g.3mg/kg Diazepam intraperitoneallyAfter 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 intraperitoneallyAfter 15 min, inject nicotine (0.2 cc of 3% intraperiotneally)

Mouse D= 16.7 gInject 5mg Dilantin intraperitoneallyAfter 15 min, inject nicotine (0.2 cc of 3% intraperiotneally)

TIME (min)

OBSERVATIONS

0 Dilatin injected

3 Slowing down, mild drowsy

5 ???

6 Little bit of convulsion – few hairs standing

9 Rapidly bitting tail

12 Moving in circle, tail bitting

13 Rapidly moving around but stopping

16 0.2 cc nicotine injected, more active

17 Rapidly bitting tail

19 Convulsing

20 Lost its balance

21 Lie on its side, unable to move one leg

23 Stop moving around but still tried to move

TIME (min)

OBSERVATIONS

25 Feet are just hanging but is able to turn

27 Few feet movement

28 Started to bite and scratch face

29 On its belly with few head movement

30 Muscle twitching on back legs

31 Rapid breathing but no movement

32 Front leg twitching

40 Still breathing

44 Still breathing, leg twitching

45 Back leg moving

46 Body twitching

50 Head is moving up

Mouse D= 16.7 gInject 5mg Dilantin intraperitoneallyAfter 15 min, inject nicotine (0.2 cc of 3% intraperiotneally)

Mouse E

• 1:06 seconds, first reaction. After the next med, 7 minutes was unresponsive

.3mg/kg Diazepam intraperitoneallyAfter 15 min, inject nicotine (0.2 cc of 3% intraperiotneally)

Mouse F:

• 0:10 seconds 1st reaction. After 6 minutes significant decrease of behavior. After 9 minutes, unresponsive

= 16.7 gInject 0.2cc of 3% NicotineThen inject 3mg/kg Diazepam during convulsions

ResultsMouse G

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

Weight= 16.8 gInjected nicotine .2cc of 3 %Then dilatin 1g

ResultsMouse G

Weight= 16.8 gInjected nicotine .2cc of 3 %Then dilatin 1g

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 anti-seizure 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 ResultsA

nicotineConvulsions Convulsions

BPhenobarbitol

nicotineSedation Seizure Sedation

CDiazepam nicotine

Sedation Seizure Recovery Seizure

DDilantin nicotine

Sedation normal activity Death

ENicotine

phenobarbitalConvulsion normal activity Death

FNicotine diazepam

Convulsion normal activity Death

G Nicotine dilantin

Convulsion normal activity Death

#4 Mechanism of Action of Nicotine

• At low to moderate doses, nicotine is a cholinergicagonist – 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

Carbamazepine

Phenobarbital

Primidone

Phenytoin

Carbamazepine

Primidone

#6 Different Types of Epilepsy and management

GeneralizedTonic-clonic(grand-mal)

Absence(petit)

Phenytoin

Carbamazepine

Phenobarbital

Primidone

Valproic Acid

Ethosuximide

Clonazepam

MycoclonicFebrile Seizures

in ChildrenStatus

Epilepticus

Valproi Acid

Valproic Acid

Clonazepam

Diazepam Phenytoin

Phenobarbital

DiazepamLorazepam

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.