This document discusses preclinical screening methods for sedative and hypnotic drugs. It describes physiology of sleep and the basic pharmacology of sedative-hypnotics. In vivo screening methods are outlined, including open field tests to measure sedation and hole board tests or combined open field tests to measure sedation and curiosity. Tests to measure hypnotic effects include potentiation of hexobarbital sleeping time and experimental insomnia in rats. In vitro screening methods like EEG registration in conscious cats and automated rat sleep analysis are also summarized. The document provides an overview of preclinical tests used to characterize new sedative and hypnotic compounds.

1. Presented by:
Syed Mubasheer Ahmed
M.Pharm. (Pharmacology),
School of Pharmacy,
S.R.T.M.University, Nanded.
Preclinical Screening of Sedative
And Hypnotics

2. CONTENT
• Introduction
• Physiology of Sleep
• Basic Pharmacology of Sedative-Hypnotics
• Chemical Classification
• Screening Method of Sedative & Hypnotic
2

3. 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)
3
Introduction:

4. Physiology of Sleep
Sleep centres:
1) Raphe nuclei in lower pons and medulla
2) Medullary synchronization area
3) Diencephalic sleep areas
4) Basal forebrain sleep area
4http://images.google.com/imgres?imgurl=brain-control-center-320

5. Phases of Sleep
1) Slow-wave sleep (NonREM):
– Phase 1-4
2) Paradoxal/desynchronized sleep:
(REM- Rapid Eye Movements)
5

6. 6

7. Sleep and EEG waves
7

8. Sleep disorders
• Insomnia
• Fatal Familial Insomnia
• Parasomnias
• Narcolepsy
• Restless leg sydrome
• Sleep paralysis
• Obstructive Sleep Apne
• Syndrome
8
(www.sinancanan.net, Physiology of Sleep)

9. 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 sedation
Graded dose-dependent depression of central nervous system
(Zhang Yanmei Sedative-Hypnotic Drugs ,Department of Pharmacology) 9

10. CHEMICAL CLASSIFICATION
Benzodiazepines:
According to uses
Anxiolytics:
Lorazepam Oxazepam Alprazolam
Chlordiazepoxide Diazepam Prazepam
Clonazepam
Hypnotics
1. short: Triazolam
2. Intermediate: Lorazepam , Estazolam
3. Long: Flurazepam, Quazepam
Preanesthetic
Diazepam-Midazolam
10

11. Barbiturates
Longacting(24-28h):Phenobarbitone
Intermediate(8-24h): Amylobarbitone
Short-acting(3-8h):
Pentobarbitone
Secobarbitone
Amobarbital
Ultrashortacting(25minutes):Thiopental
Miscellaneous: ( non BDZ non barbiturate drugs).
Zolpidem
Zaleplon
(K.D. Tripathi, Essentials of medical pharmacology 5th edition, page no.357&358) 11

12. 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 ) 12

13. Preclinical Screening Method of Sedative &
Hypnotic
1. In vivo Method
2. In vitro Method
In vivo Method
For Sedative Activity
1. Open field test
2. Hole-board test
3. Combined open field test
4. EEG analysis from rat brain by telemetry
(Vogel , drug discovery and evaluation : pharmacological assay , 2nd edition page no.495) 13

14. 1. Open Field Test:
 Purpose and Rationale:
• Rats and Mice are used.
• To Interrupt the light beams as a measure of movements.
• “Open Field”
14

15. 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.
15

16.  Evaluation:
• Dose Response Curves are obtained.
• Various parameters show different results.
• Statistical Comparison of various doses is possible.
16

17. 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
17

18. 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.
18

19.  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.
19

20. 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.
20

21. 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.
21

22. 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.
22

23. 23
1. Potentiation of Hexobarbital sleeping time
2. Experimental insomnia in rats
3. EEG registration in concious cats
4. Automated rat sleep analysis system
(Vogel , drug discovery and evaluation : pharmacological assay , 2nd edition page no.495)
2) In vivo Method
For Hypnotic activity:

24. 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.
24

25.  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
25

26.  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.
26

27. 27
2. Experimental insomnia in rats:
• Purpose & Rationale:
James & Piper(1978)
Potential hypnotic compounds in rats

28.  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.
EVALUATION
The sleep- wake cycle is definitely altered by the stress
procedure.
For screening procedure, the method is too expensive &
time-consuming.
28

29. 29
3. EEG registration in conscious cats:
Purpose & Rationale
The effect of hypnotic on sleep pattern of EEG tracings

30.  Procedure:
Female cats weighing2.5-3.5kg are anesthetized & prepared with
bipolar subcortical electrodes in the reticular formation
Cats 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
30

31. 4 . Automated rat sleep analysis system
 Purpose & Rationale:
Ruigt et al.(1988a,b,1993)
which allows classification of psychotropic drugs
Records & analyzes bioelectrical signals
31

32.  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.
32

33. 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
33

34. 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)
34

35. 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.
35

36. REFERENCES
36
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.

37. 37