Presented by:Syed Mubasheer AhmedM.Pharm. (Pharmacology),School of Pharmacy,S.R.T.M.University, Nanded.

Preclinical Screening of Sedative And Hypnotics

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CONTENT

• Introduction• Physiology of Sleep• Basic Pharmacology of Sedative-Hypnotics• Chemical Classification• Screening Method of Sedative & Hypnotic

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Sedation is defined as subjective feelings of drowsiness and sleepiness, and also as objectively measured slowing of psychomotor functioning.

Induce central nervous system (CNS) depression, by enhancing the function of GABA-mediated chloride channels via agonism at the GABA receptor containing the α1 subunit.

(David C. Lee and Kathy Lynn Ferguson, Sedative-Hypnotics,1060)

Introduction:

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Physiology of Sleep

Sleep centres:1) Raphe nuclei in lower pons and medulla2) Medullary synchronization area3) Diencephalic sleep areas4) Basal forebrain sleep area

http://images.google.com/imgres?imgurl=brain-control-center-320

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Phases of Sleep

1) Slow-wave sleep (NonREM):

– Phase 1-4

2) Paradoxal/desynchronized sleep:

(REM- Rapid Eye Movements)

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Sleep and EEG waves

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Sleep disorders• Insomnia• Fatal Familial Insomnia• Parasomnias• Narcolepsy• Restless leg sydrome• Sleep paralysis• Obstructive Sleep Apne• Syndrome

(www.sinancanan.net, Physiology of Sleep)

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BASIC PHARMACOLOGY OF SEDATIVE-HYPNOTICS

Reduce anxiety and exert a calming effect with little or no effect on motor or mental functions. Hypnotic drug should produce drowsiness Involve more pronounced depression of the central nervous system than sedationGraded dose-dependent depression of central nervous system

(Zhang Yanmei Sedative-Hypnotic Drugs ,Department of Pharmacology)

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CHEMICAL CLASSIFICATION

Benzodiazepines:According to uses

Anxiolytics: Lorazepam Oxazepam Alprazolam Chlordiazepoxide Diazepam Prazepam ClonazepamHypnotics

1. short: Triazolam 2. Intermediate: Lorazepam , Estazolam3. Long: Flurazepam, Quazepam

Preanesthetic Diazepam-Midazolam

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Barbiturates Longacting(24-28h):Phenobarbitone Intermediate(8-24h): Amylobarbitone Short-acting(3-8h):

PentobarbitoneSecobarbitoneAmobarbital

Ultrashortacting(25minutes):Thiopental

Miscellaneous: ( non BDZ non barbiturate drugs).

Zolpidem

Zaleplon

(K.D. Tripathi, Essentials of medical pharmacology 5 th edition, page no.357&358)

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What are their mechanisms? Act on GABA receptors, either increasing or decreasing the concentrations and activities of the neurotransmitters, like GABA, serotonin, noradrenaline, thus inducing sleep.

(http://images.google.com/imgres?imgurl=http://www.long-island-hypnotherapy.com/images/anxiety )

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Preclinical Screening Method of Sedative & Hypnotic

1. In vivo Method2. In vitro Method

In vivo MethodFor Sedative Activity

3. Open field test4. Hole-board test5. Combined open field test6. EEG analysis from rat brain by telemetry

(Vogel , drug discovery and evaluation : pharmacological assay , 2nd edition page no.495)

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1. Open Field Test:

Purpose and Rationale:• Rats and Mice are used.• To Interrupt the light beams as a measure of movements.• “Open Field”

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Procedure

• square open field arena • 2 rows of 8 photocells, sensitive to infrared light,• Measurements are made in the dark in a ventilated, sound-

attenuating box.

• Motor activity: All interruptions of photo beams in the lower rows.

• Peripheral motor activity: Activation of photo beams in the lower rows, photo-beams spaced 25 mm from the wall were also activated.

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Evaluation:• Dose Response Curves are obtained.• Various parameters show different results.• Statistical Comparison of various doses is possible.

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2. Hole-board Test

Purpose and Rationale:• To Evaluate behavior of mice such as curiosity and exploration.• Open field with holes on the bottom.• The “planche à trous” or “hole-board”• The hole-board has a size of 40 × 40 cm

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Procedure:

• NMRI strain Mice of either sex weighing between 18 and 22 gm are used.

• Sixteen holes with a diameter of 3 cm each are distributed evenly on the floor.

• The board is elevated so that the mouse poking its nose into the hole does not see the bottom. Nose-poking is measured by visual observation counted by electronic devices in more recent modifications.

• Usually, 6 animals are used for each dose and for controls. • Thirty minutes after administration of the test compound the first

animal is placed on the hole-board and tested for 5 min.

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Calculation: • The number of counts for nose-poking of treated animals is

calculated as percentage of control animals. Evaluation:• Poking the nose into a hole is a typical behavior of mice indicating

a certain degree of curiosity. • Evaluation of this component of behavior has been proven to be

quite useful. • Benzodiazepines tend to suppress nose pocking at relatively low

doses.

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3. Combined open field test

Purpose And Rationale:

• To determine the locomotion and curiosity by using a modification of Hole-Board Test.

• Several equipments of such type are available commercially now.

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Procedure:• NMRI-strain Male mice with an average weight of 30 g are used. • Each animal is tested individually in an automated open-field box which

consists of a black Plexiglas cage (35 × 35 × 20 cm) with a post (8 × 8 × 20 cm) in the center of the cage.

• Two evenly spaced photo cell beams perpendicular to the wall and 2 cm above the floor divide the box into 4 compartments. Every photo cell beam interruption is registered automatically as an activity count.

• A row of 4 photocell beams is mounted 1 cm outside of the holes and automatically records every exploratory nose-poke.

• 30 min. after intraperitoneal and 60 min. after oral administration of the test compound the animal is placed into the cage and the behavior recorded for a period of 5 min.

• Ten mice are used for each dose as well as for controls.

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Evaluation:

• Counts for motility (interruption of photo cell beams inside the cage) and for curiosity (interruption of photo cell beams outside the cage due to nose-poking) are recorded individually.

• The mean values of the treated groups are expressed as percentage of the control group.

• Using different doses, dose-response curves can be obtained.

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1. Potentiation of Hexobarbital sleeping time2. Experimental insomnia in rats3. EEG registration in concious cats4. Automated rat sleep analysis system

(Vogel , drug discovery and evaluation : pharmacological assay , 2nd edition page no.495)

2) In vivo MethodFor Hypnotic activity:

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1) Potentiation of Hexobarbital sleeping time

Purpose And Rationale:

• To elucidate CNS- active properties of drugs• The loss of righting reflex is measured as criterion for the

duration of hexobarbital-induced sleeping time.• Mice are used.

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Procedure:

•10 male NMRI-mice are used

•Avg.wt-18-22g

•60mg/kg hexobarbital injected i.v.

•Duration of righting reflex is measured

EVALUATION:

•The percent change in duration of anesthesia is calculated

•ED50 values can be calculated

CRITICAL ASSESSMENT OF THE METHOD

•The anxiolytic agent of the benzodiazepine type show an uniform

pattern with oral ED50 Values of less than 1mg/kg.

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2. Experimental insomnia in rats:• Purpose & Rationale:

James & Piper(1978)

Potential hypnotic compounds in rats

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Procedure:Male Wistar rats(200-275g) are prepared for chronic electroencephalographic & electromyographic recording. The animals are placed into sound-attenuated recording chambers with grid floors. Animals are exposed to electric footshocks for 8 h.in the form of a 0.5mA pulse of 15ms width for 30s at 1Hz.

EVALUATIONThe sleep- wake cycle is definitely altered by the stress procedure.For screening procedure, the method is too expensive & time-consuming.

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3. EEG registration in conscious cats: Purpose & Rationale The effect of hypnotic on sleep pattern of EEG tracings

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Procedure:Female cats weighing2.5-3.5kg are anesthetized & prepared with bipolar subcortical electrodes in the reticular formationCats are taken on experimental chamber 70×80×80cm high.Continuous recording for up to 96 h are amplified & stored in a recorder

EVALUATION:The data are analyzed of variance with subjects days & drug as factors

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4 . Automated rat sleep analysis system

Purpose & Rationale:Ruigt et al.(1988a,b,1993) which allows classification of psychotropic drugs Records & analyzes bioelectrical signals

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Procedure:

Epidural screw electrodes are implanted over the parieto-occipital cortex of male rats weighing 250–300 g for the recording of EEG against a frontal electrode.

Stainless-steel wire electrodes are inserted in the dorsal neck musculature for recording the electromyogram

The discriminant function is derived from a discriminant analysis of visually classified representative recording segments from different sleep stages recorded during a separate calibration experiment for each rat.

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Six sleep-wake stages are distinguished

including2 waking stages:

(1) active waking

(2) quiet waking without movement.

(3) quiet sleep,

(4) deep slow-wave sleep

(5) pre-REM sleep

(6) REM sleep

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EVALUATION Fairchild et al. (1969, 1971,1975). Sleep stage-dependent and sleep-independent parts of the EEG power spectrum Normal canonical discriminant analysis is done on EEG frequency bands (1–3, 3–6,6–9, 9.5–20Hz) from representative segments of only visally classified sleep stages (quiet waking, deepsleep and REM sleep)

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CRITICAL ASSESSMENT OF THE METHOD

Antidepressants,antipsychotics and stimulants can be discriminated from each other and from placebo successfully from each other and from placebo by this method Nootropics classified as placebo.

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REFERENCES

1. Vogel Drug discovery and evaluation : pharmacological assay, 2nd edition page no.395-399 & 495-499

2. K.D. Tripathi, Essentials of medical pharmacology 6th edition, page no.388-400)

3. www.sinancanan.net Physiology of Sleep

4. Zhang Yanmei Sedative-Hypnotic Drugs ,Department of Pharmacology.

5. http://images.google.com/imgres?imgurl=http://www.long-island-hypnotherapy.com/images/anxiety Accessed May2006

6. David C. Lee and Kathy Lynn Ferguson, Sedative-Hypnotics,1060.

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Thank You