Presentation on theme: "Experiment #1 The Effects of Anticonvulsant Agents on Mice Group 1 and 2 Pharmacology B December 12, 2006."— Presentation transcript:
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 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 PredicablePredictableNot 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 PredictableNot 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… PredictableNot PredictableNot 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 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.Antagonism of serotonin 2.Increased release of and/or facilitation of gamma- aminobutyric acid (GABA) activity 3.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
Procedure All mice were injected intraperitoneally with the specific drug in the table below. MouseInitiallyAfter 15 minutes A0.2cc of 3% NicotineNothing B 1mg Phenobarbital Sodium 0.2cc of 3% Nicotine C.3mg/kg Diazepam 0.2cc of 3% Nicotine D5mg Dilantin0.2cc of 3% Nicotine E Phenobarbital - During convulsions F0.2cc of 3% Nicotine.3mg/kg Diazepam - During convulsions G0.2cc 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
= 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) TIMEOBSERVATION 00.05 cc diazepam injected (dose 0.05cc / 20 gm) 15 min0.2 cc nicotine injected 16 min 26 secEvidence of seizure by hair standing & whiskers straight, minimal movement but still on all fours 17 min 20 secPrior observations, straight tail, no foot twitching 27 min 35 secPrior observations & foot twitching 34 minMouse is more active exhibited by standing & walking 35 minMouse lies down but still showing prior evidence of seizures 36 minMouse 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)
Mouse D = 16.7 g Inject 5mg Dilantin intraperitoneally After 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 g Inject 5mg Dilantin intraperitoneally After 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 intraperitoneally After 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 g Inject 0.2cc of 3% Nicotine Then inject 3mg/kg Diazepam during convulsions
Results Mouse G MinActions 0 minInjected nicotine 1 min 20 secWent into convulsion 1 min 21 secInjected dilatin 2 min 55 secHeavy and rapid breathing rate lying down no mvt 5 minHeavy and rapid breathing rate no movement 12 minNormal deep breathing rate no mvt 15min 34 secNormal breathing rate lying on its side 17 min 12 secSlow breathing rate no mvt 22 min 45 secStopped breathing completely--- dead Weight= 16.8 g Injected nicotine.2cc of 3 % Then dilatin 1g
Results Mouse G Weight= 16.8 g Injected 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 MouseExpected ResultsActual Results A nicotine Convulsions B Phenobarbitol nicotine Sedation SeizureSedation C Diazepam nicotine SedationSeizure Recovery Seizure D Dilantin nicotine Sedation normal activityDeath E Nicotine phenobarbital Convulsion normal activityDeath F Nicotine diazepam Convulsion normal activityDeath G Nicotine dilantin Convulsion normal activityDeath
#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 GABA A receptor. Increase frequency of Cl - channel opening. ※ GABA GABA A – CNS GABA B – 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 Phenytoin Carbamazepine Phenobarbital Primidone Complex Phenytoin Carbamazepine Primidone
#6 Different Types of Epilepsy and management Generalized Tonic-clonic (grand-mal) Phenytoin Carbamazepine Phenobarbital Primidone Valproi Acid Absence (petit) Valproic Acid Ethosuximide Clonazepam Mycoclonic Valproic Acid Clonazepam Febrile Seizures in Children Diazepam Status Epilepticus Phenytoin Phenobarbital Diazepam Lorazepam
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.LD50ratsmicecocaine
Diagnosing Increased nicotine or cotinine (the nicotine metabolite) is detected in urine or blood, or increased serum nicotine levels occur.cotinine metabolite
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.insecticidechildrenemergency roomcigarettescigarette buttsnicotineadult
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.