DRUG SCREENING OF

HYPNOTIC ACTIVITY

CONTENTS

• Physiology of Sleep

• Sedative-hypnotics

• Screening of hypnotic activity

PHYSIOLOGY OF SLEEP

SLEEP

• Sleep Unconsciousness from which a person can be aroused by

sensory or other stimuli.

• Coma

• 2 stages of sleep• Slow-wave sleep/ Non Rapid Eye Movement (NREM) sleep

• Rapid Eye Movement (REM) sleep/ Paradoxical Sleep

BRAIN WAVES• Electroencephalogram

• German psychiatrist, Hans Berger

• Brain wave intensity from scalp surface 0-200 microvolts

• Frequency once in few seconds to 50 or more per second.

• Character of wave depends on• degree of activity in respective parts of the cerebral cortex

• States of wakefulness, sleep & coma.

BRAIN WAVES

• In normal healthy individual EEG waves can be classified as,

•Alpha

• Beta

• Theta

•Delta

ALPHA WAVES• Rhythmical waves with frequency 8-13 cycles

• Present in normal adults while awake.

• Origin• Spontaneous feedback oscillation in diffuse thalamocortical system,

including the reticular activating system in the brain stem as well.

• Intensity 50 microvolts

• Disappear during sleep.

BETA & THETA WAVES• BETA WAVES• Frequency 14-80 cycles per second• Recorded from parietal and frontal regions

• THETA WAVES• Frequency 4-7 cycles per second• Origin• Parietal and temporal regions in children• Emotional stress in adults frustration and disappointment• Degenerative disorders

DELTA WAVES• Includes all waves of EEG with frequency less than 3.5 cycles per

second.

• Intensity voltage greater than 3-4 times the other waves

• Origin• Deep sleep in infancy• Serious organic brain disease• In cortex of animals that have had subcortical transections separating the

cerebral cortex from the thalamus.

NEUROCHEMICAL MECHANISMS PROMOTING SLEEP & WAKEFULNESS

• Circadian rhythm 8 h sleep; 16 h wakefulness

• Nuclei in brain stem as well as hypothalamus

• Electrical stimulation of the posterior hypothalamus wakefulness

• Electrical stimulation of anterior hypothalamus & adjacent basal

forebrain sleep

NEUROCHEMICAL MECHANISMS PROMOTING SLEEP & WAKEFULNESS

• Brainstem RAS norepinephrine, serotonin, or acetylcholine.

• Forebrain neurons• preoptic neurons in hypothalamus GABA

• Posterior hypothalamic neurons histamine.

STAGES OF SLEEP

• Slow wave/ NREM sleep

• REM/ paradoxical/ Desynchronized sleep

SLOW-WAVE SLEEP

• Deep, restful sleep a person experiences after being awake for many

hours

• Strong brain waves; low frequency

• Divided into 4 stages

SLOW-WAVE SLEEP

REM SLEEP

• 25% total sleep time.

• Recurs every 90 minutes; Lasting 5-30 min.

• Not restful; Associated with vivid dreaming

• Irregular, high frequency waves desynchronized nervous activity.

REM SLEEP- FEATURES

• Active dreaming & bodily muscle movements

• Difficult to arouse by sensory stimuli compared to NREM sleep

• Muscle tone depressed

• Heart rate, Respiratory rate Irregular Dream state

REM SLEEP- FEATURES• Brain is highly active; Metabolism increased by 20%

• EEG brain wave pattern similar to that occurring during wakefulness.

• Paradoxical sleep a paradox that a person can still be asleep despite

marked activity in the brain.

SEDATIVE- HYPNOTICS

SEDATIVE- HYPNOTICS

• Sedation decreased responsiveness to any level of stimulation.

• Sedative reduces excitement; calms subject without inducing sleep.

• Hypnotic induces sleep, and/or maintains sleep.

• Similar to normal sleep

• In animal expt, applied to a much deeper stage of central depression of drug

induced unconsciousness associated with loss of muscle tone & righting reflex.

SEDATIVE- HYPNOTICS

• Global CNS depressants

• Sedatives Slow acting; Flat DRC

• Hypnotics Rapid onset, Short duration, Steep DRC

SEDATIVE- HYPNOTICS- CLASSIFICATION

1. BARBITURATES

• LONG ACTING• Phenobarbitone

• SHORT ACTING• Butobarbitone• Pentobarbitone

• ULTRA-SHORT ACTING• Thiopentone• Methohexitone

SEDATIVE- HYPNOTICS- CLASSIFICATION

2. BENZODIAZEPINES

3. Newer Nonbenzodiazepine hypnotics Zopiclone, Zolpidem, Zaleplon.

• HYPNOTIC• Diazepam• Flurazepam• Nitrazepam• Alprazolam• Temazepam• Triazolam

• ANTIANXIETY• Diazepam• Chlordiazepoxide• Oxazepam• Lorazepam• Alprazolam

• ANTICONVULSANT• Diazepam• Lorazepam• Clonazepam• Clobazam

MECHANISM OF ACTION

EFFECT AS A HYPNOTIC

• Latency of sleep onset is decreased

• Duration of Stage 2 NREM sleep is increased

• Duration of REM and stage 4 NREM sleep is decreased

DRUG SCREENING

DRUG SCREENING FOR HYPNOTIC ACTIVITY

• In vivo methods

• Potentiation of Hexobarbital Sleeping time

• Experimental Insomnia in rats

• EEG registration in conscious cats

• Automated Rat Sleep Analysis System

POTENTIATION OF HEXOBARBITAL SLEEPING TIME

• PURPOSE & RATIONALE

• To elucidate CNS-active properties of a drug

• Main criteria loss of righting reflex

• Mice are used metabolic elimination of hexobaribtal is rapid

POTENTIATION OF HEXOBARBITAL SLEEPING TIME- PROCEDURE

• Inclusion criteria 10 male NMRI mice; Avg weight- 18-22g

• Administer test compound/ reference standard (3mg/kg diazepam) oral; IP; SC

• Hexobarbital- 60mg/kg- IV:

• 30 min after IP or SC injection

• 60 min after oral

POTENTIATION OF HEXOBARBITAL SLEEPING TIME- PROCEDURE

• Animals are placed on their backs on a warm pad- 37 ͦ C

• Duration of loss of righting reflex is measured until its regained

• Inj. Hexobarbital causes anaesthesia for 15 minutes.

POTENTIATION OF HEXOBARBITAL SLEEPING TIME

• EVALUATION• Mean values of duration of anesthesia- recorded• % change • ED 50 calculated

• CRITICAL ASSESSMENT• Anxiolytic agents of BZD type uniform pattern with oral ED 50 < 1mg/kg• Neuroleptics haloperidol, chlorpromazine• Test unspecific

POTENTIATION OF HEXOBARBITAL SLEEPING TIME- OTHER USES

• To reduce hexobarbital sleeping time

• Pentylenetetrazol; Methamphetamine; Aminophylline

• Repeated administration induction of metabolic enzymes increased

destruction of hexobarbital reduced sleeping time.

MODIFICATIONS OF THE TEST

• Hexabarbital 25mg/kg Thiopental IV

• Interaction of various psychotropic agents with sleep induced by barbital (180 mg/kg) or pentobarbital (50 mg/kg) i.p. in mice. (1)

• Hexabarbital Barbital-Na Barbital is not biotransformed by liver microsomal system. (2)

1. Simon P, Chermat R, Doaré L, Bourin M, Farinotti R (1982) Interactions imprévues de divers psychotropes avec les effets du barbital et du pentobarbital chez la souris. J Pharmacol (Paris) 13:241–252

2. Fujimori H (1965) Potentiation of barbital hypnosis as an evaluation method for central nervous system depressants. Psychopharmacologia 7:374–378

EXPERIMENTAL INSOMNIA IN RATS

• PURPOSE & RATIONALE

• James & Piper 1978 evaluation of potential hypnotic compounds in rats

• Foot-shock induced insomnia in rats suitable model for insomnia

• INCLUSION CRITERIA

• Male Wistar Rats

• Weight 200-275 g

EXPERIMENTAL INSOMNIA IN RATS- PROCEDURE• Rats are prepared for chronic electroencephalographic & electromyographic

recordings.

• Four silver/silver chloride epidural electrodes and two disc nuchal electrodes are implanted.

• 10 days for recovery

• Placed into sound attenuated recording chambers with grid floors.

• The frontal-occipital electroencephalogram and the electromyogram are recorded via nonrestraining recording leads on a polygraph and a tape recorder.

EXPERIMENTAL INSOMNIA IN RATS- PROCEDURE

• Day 1 Administer vehicle & control nonstress recording for 8h

• Day 2 Administer vehicle; Expose to foot shocks for 8h• Foot-shock grid floor of recording chamber • 0.5mA pulse of 15 ms width for 30 s at 1Hz.• EEG and EMG recording circuits are automatically interrupted. • Thus, each shock period of 30 s is followed by an interval of 30min.

• Day 3 Administer test compound/ standard recordings obtained during shock sessions for 8 h.

EXPERIMENTAL INSOMNIA IN RATS

• EVALUATION

• Stress procedure alteration of sleep-wake cycle

• Increased wakefulness & Stage I NREM

• Decreased NREM II & REM

• Phenobarbital & BZD antagonize these changes partially

• CRITICAL ASSESSMENT• Too expensive & time consuming

MODIFICATIONS IN THE METHOD

• Shortened-protocol 2.5 h non-stressed control period administration of

drug/vehicle 5.5-h recording of the electrocorticogram in the presence of

foot shock. (1)

• The effect of anxiolytic and hypnotic drugs on sleep circadian rhythms in

the rat. (2)

1. Gardner CR, James V (1987) Activity of some benzodiazepine receptor ligands with reduced sedative and muscle relaxantproperties on stress-induced electrocorticogram arousal in sleeping rats. J Pharmacol Meth 18:47–542. Laval J, Stenger A, Briley M (1991) Effect of anxiolytic and hypnotic drugs on sleep circadian rhythms in the rat. In: Briley M, File SE (eds) New Concepts in Anxiety. McMillan Press Ltd., London, pp 338–346

EEG REGISTRATION IN CONSCIOUS CATS

• PURPOSE AND RATIONALE

• Effect of hypnotics on sleep pattern of EEG tracings in conscious

freely moving cats with chronically implanted electrodes.

• Inclusion Criteria female cats weighing 2.5-3.5 kg

EEG REGISTRATION IN CONSCIOUS CATS- PROCEDURE

• Animals prepared with bipolar subcortical electrodes in the reticular formation, dorsal hippocampus, either amygdala/ caudate nucleus

• Cortical screw electrodes are placed over the anterior suprasylvian, lateral, medial suprasylvian and ectosylvian gyri.

• Two Teflon coated steel wires are placed in the cervical neck muscles.

• All wires are connected to a subminiature socket and implanted in dental acrylic.

EEG REGISTRATION IN CONSCIOUS CATS- PROCEDURE

• Experimental chamber 70X80X80 cm high

• Cat is connected to a cable exits through top center of cage through mercury swivel.

• Recordings amplified and stored upto 96h.

• Recordings of the cortical EEG, cervical neck muscle tone and reticular formation multiple unit activity are obtained.

• Recordings analyzed for REM, NREM and wakefulness.

MODIFICATION OF THE METHODS

• EEG & EMG Electro-oculogram

• Evaluation of EEG recordings in freely moving rats by visual analysis for wakefulness, slow wave sleep or paradoxical sleep.

AUTOMATED RAT SLEEP ANALYSIS SYSTEM

• PURPOSE AND RATIONALE• Described by Ruigt et.al 1988

• Allows classification of psychotropic drugs such as potential antidepressants, antipsychotics and stimulants

• Involves recording and analysis of bioelectrical signals from several animals over extended periods of time.

• Analysis based on 3 signals the parietooccipital EEG, nuchal EMG and a movement indicator signal.

AUTOMATED RAT SLEEP ANALYSIS SYSTEM- PROCEDURE

• EEG Epidural Screw electrodes Parieto-occipital cortex

• EMG Stainless steel wire electrodes dorsal neck musculature

• 29 h recordings in sound attenuated Faraday cages.

• An open-ended wire of non-restraining flat cable connects rats to swivel commutator, amplification units & A/D conversion units.

AUTOMATED RAT SLEEP ANALYSIS SYSTEM- PROCEDURE

• This is connected to minicomputer system for online spectral EEG analysis & data compression.

• Off-line sleep staging noted- based on 5 spectral EEG band values.

• 6 sleep-wake stages distinguished

AUTOMATED RAT SLEEP ANALYSIS SYSTEM- PROCEDURE

• 32 rats 4 groups

• Drug administration beginning of light cycle

• After each expt 2-3 weeks allowed for washed out.

• Drug effect on sleep-waking behavior assessed.

• Parameters percentage time spent in each of the sleep stages per 30-min period and per rat extracted from hypnogram.

AUTOMATED RAT SLEEP ANALYSIS SYSTEM- EVALUATION

1. Active waking characterized by movement, theta activity and high EMG

2. Quiet waking without movement.

3. Quiet sleep, characterized by EEG spindles.

4. Deep slow-wave sleep with prominent delta activity.

5. Pre-REM sleep with spindles against a background of theta activity and low

EMG

6. REM sleep with theta activity and low EMG.

REFERENCES

• Textbook of Medical Physiology, Guyton, 11th edition

• Ganong’s review of medical physiology, 23rd edition.

• Goodman and Gillman Manual of Pharmacology and therapeutics 12th edition

• Essentials of Medical Pharmacology, K. D. Tripathi, 6th edition

• Drug Discovery and Evaluation of Pharmacological Assays- H. Gerhard. Vogel- 3rd edition

THANK YOU