Experiment #1 The Effects of Anticonvulsant Agents on Mice

1. Experiment #1The Effects of Anticonvulsant Agents on Mice Group 1 and 2 Pharmacology B December 12, 2006

2. 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

3. 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.

4. Mechanism of actionand anticonvulsanteffect of the three drugs

5. 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.

6. 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

7. 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

8. Phenobarbital Drug Interactions • Barbiturates • Induce hepatic microsomal drug metabolizing enzymes • Additive central nervous system depression with other central nervous system depressants

9. 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.

10. 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)

11. 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.

12. Dilantin Drug Interactions • Induces hepatic microsomal drug metabolism • Susceptible to inhibition of metabolism by CYP2C9 and to a lesser extent CYP2C19

13. Dilantin Drug Interactions cont…

14. 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.

15. 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. • Antagonism of serotonin • Increased release of and/or facilitation of gamma-aminobutyric acid (GABA) activity • Diminished release or turnover of acetylcholine in the CNS ↓ Inhibit generation of action potential

16. 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.

17. 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

18. 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

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

20. Parameters

21. Mouse A = 15.5 g Inject 0.2cc of 3% Nicotine intraperitoneally

22. Mouse A = 15.5 g Inject 0.2cc of 3% Nicotine intraperitoneally

23. Mouse B

24. 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)

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

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

27. Mouse D = 16.7 g Inject 5mg Dilantin intraperitoneally After 15 min, inject nicotine (0.2 cc of 3% intraperiotneally)

28. Mouse D = 16.7 g Inject 5mg Dilantin intraperitoneally After 15 min, inject nicotine (0.2 cc of 3% intraperiotneally)

29. 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

30. Mouse F: = 16.7 g Inject 0.2cc of 3% NicotineThen inject 3mg/kg Diazepam during convulsions • 0:10 seconds 1st reaction. After 6 minutes significant decrease of behavior. After 9 minutes, unresponsive

31. ResultsMouse G Weight= 16.8 g Injected nicotine.2cc of 3 % Then dilatin 1g

32. ResultsMouse G Weight= 16.8 g Injected nicotine.2cc of 3 % Then dilatin 1g

33. 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

34. #4 Discussion of Results of Experiment

35. #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.

36. Can dianepam, phenobarbital and dilantin act as a prophylaxis against nicotine? Why or why not?

38. 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

39. 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

40. 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

41. 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.

42. 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.

43. #6 Different Types of Epilepsy and management

44. #6 Different Types of Epilepsy and management

45. Give the conclusion of the experiment

46. 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.

47. Symptoms • vomiting and nausea, diarrhea • headaches • difficulty breathing • palpitations • stomach pains/cramps • seizures • weakness • increased drooling

48. Diagnosing • Increased nicotine or cotinine (the nicotine metabolite) is detected in urine or blood, or increased serum nicotine levels occur.

49. 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.

50. 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.