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PHR BIOEVALUATION DRUG SCREENING Bioevaluation of drugs Powered By Docstoc
					              PHR 582

Bioevaluation of drugs acting on the

          Dr. Rowaida Refaat
Intended learning outcomes (ILOs):

By the end of this lecture students should be able to:

  1- Understand the different screening tests for antipsychotic drugs.
  2- Compare between the screening tests for narcotic and non-narcotic
  3- Discuss the different methods of induction of experimental epilepsy
     and determine their value in screening of antiepileptic drugs.
  4- Recognize the in vitro and in vivo assays of antidepressants.

          Screening of drugs acting on the central nervous system

Drugs acting on the CNS include:

   Central nervous system depressants:
CNS depressants include sedatives & hypnotics and anesthetics.

   Screening tests for CNS depressants
   a) Loss of reflexes
    Righting reflex (the animal cannot remain on its back)
    Corneal reflex (movement of the eyelid or cornea)
The drug under test (CNS depressant) is injected into rats, then after
30-60 min, both reflexes are examined. If the animal remains on its back for
30 seconds, then this is a positive loss of righting reflex.

   b) Antagonism of CNS stimulants
Control rats are injected with CNS stimulants e.g. nicotine or
pentylenetetrazole which produce convulsions and tremors.
Another group of rats is injected with the drug under test, then after one
hour injected with entylenetetrazole. If protection from convulsions occurs,
then the drug is a CNS depressant.

   c) Potentiation of the sleeping time of barbiturates
CNS depressants potentiate the sleeping time of hexobarbital from 30 min
to one hour.

    Antipsychotics: (major tranquilizers)
These drugs are used to relieve disturbed schizophrenic patients
regardless of the underlying psychopathology e.g. Chlorpromazine

      Screening tests for antipsychotics:

I-      Tests depending on the change in the animal behavior

     a) Conditioned avoidance test:
      A rat is put in a wooden box except for an area of the floor, which is
      formed of stainless steel. At the top of the box, there is a lamp or a bell.
     Applying an electric shock to the stainless steel part of the floor will
     cause the rat to jump to the safe area. This is an unconditioned
     avoidance response, BUT if we turn on the light or ring the bell
     30 seconds before the electric shock, the animal will be trained so that
     when seeing the light or hearing the bell, it will avoid the punishment and
     go to the safe area before the electric shock begins. This is a
     conditioned avoidance response.

     After training the animal, the drug under test is injected I.P. and the
     conditioned avoidance response is observed after 30, 60, 120 and 180
     - When the light is turned on, the animal does not go to the safe area
        as before.
     - When the electric shock occurs without previous alarm, the animal
        goes to the safe area.
     - Therefore, tranquilizers do not impair the unconditioned avoidance
        response BUT impair the conditioned response.
     - CNS depressants impair both conditioned and unconditioned
     - A dose-response curve is plotted and the ED50 is calculated.

     b) Spontaneous hostility:

 Taming effect
Tranquilizers will lead to the following effects:
   a- Handling a monkey in a quiet room by an operator will be smoother.
   b- Animals like mice/rats and cats, or dogs and cats do not fight
  Group excitement test
Male mice are kept at 23-26ºC and divided into 3 groups each of 10-20
- Control group
- Group treated with 25mg/kg amphetamine
- Group treated with both amphetamine and a tranquilizer
The number of dead mice is counted after 20 hours and the LD50 is
determined. Tranquilizers will decrease amphetamine toxicity.

   c) Classical Pavlovian conditioning:
This test depends on experimental neurosis in dogs. If the dog sees a
spherical disc, it is given food, but if the disc is oval, the dog is deprived of
food. By training, salivation is seen only with spherical discs. Tranquilizers
prevent neurosis and no salivation is seen even with spherical discs.

    d) Induced aggressiveness:
   Male mice are isolated for several weeks. If a non-isolated mouse is
    introduced, it will be attacked within 5 minutes.
   Territorial aggression model: male and female mice or rats are
    housed together for sometime (resembles natural situation in home).
    When a foreign male is introduced, the male will fight for his territory.
    Aggression of lactating females.
   Aggression can be induced between 2 mice or rats by electricity or tail
    pinching. Antipsychotics reduce the amount of aggressive behavior.

II-   Tests depending on CNS depression
Tranquilization is the first symptom of all CNS depressants. These tests are
also used for screening all CNS depressants.

  a) Rotating rod test (Rotarod):
 A metallic rod is set to rotate at a rate of 12 rotations/ minute.

 A mouse is put on this rotating rod. Normally, the mouse can remain on
  the rod for 5 minutes.
 The drug under test is injected and the test is repeated after 30, 60, 120
  and 180 minutes. If the animal fails to remain on the rotating rod and
  falls (due to CNS depression, loss of orientation and muscular
  relaxation), then the test is considered positive and the drug is a
  tranquilizer. Determine the falling time.

   b) Chimney test:
A mouse is introduced into one end of a glass tube with an inner diameter
of 28 mm and is gently pushed from behind with a cotton padded rod
towards the opposite end while the tube is turned vertically onto a table top
with the mouse head down. Control animals easily climb backwards up to a
height of 18 cm (mark) within 30 seconds. Measure the backward climbing
time for each animal ten minutes after drug administration.

III-   Tests depending on potentiation of CNS depressants
   a) Increase in the sleeping time of barbiturates:
 Rats are injected with an ultrashort acting barbiturate “hexobarbital” and
  the sleeping time is calculated. (time elapsed from loss till recovery of
  righting reflex).
 Another group of rats is pretreated with drug (X), then after 1 hour a
   dose of hexobarbital is given to produce CNS depression & sleep.
 If the sleeping time of the drug treated group is increased more than
   the control group, then (X) is a tranquilizer.

  b) Potentiation of the analgesic effect of morphine:
 A group of rats is subjected to the hot plate test. Morphine is given
  followed by recording the reaction time.
 Another group of rats is pretreated with drug (X) and one hour later,
  morphine is given and the reaction time is recorded. If (X) is a
  tranquilizer, the effect of morphine is potentiated and the reaction time
  is increased.

IV-  Screening test depending on induction of hypothermia
     (specific test)
 Tranquilizers lower the body temperature by acting centrally on the heat
  regulating center in the hypothalamus.

 In a group of rabbits, the normal body temperature is recorded by a
  probe in the rectum (normally between 37-38°C).
 Drug (X) is injected and the body temperature is recorded after 30, 60,
  120 and 180 minutes. If the body temperature is lowered, then the drug
  is a tranquilizer. Injection of chlorpromazine (8 mg/kg) into a rabbit will
  decrease its body temperature by 0.5-2ºC.

   Anxiolytics (minor tranquilizers)

Example of these drugs are lorazepam & buspirone

   Screening tests for anxiolytics

   a) Conflict test
A rat is trained to press a bar repeatedly to obtain food pellets. A conflict
element is then introduced at intervals, indicated by an auditory signal.
Bar pressing results in an electric shock (punishment) in addition to the
food pellet (reward).
Normally, the rat stops pressing the bar (behavioral inhibition) to avoid the
shock during the period when the signal is sounding. Anxiolytic drugs
cause the rat to continue bar pressing for reward in spite of the
punishment. Other psychotropic drugs or analgesics are not effective.

   b) Aggressiveness inhibition test
Aggressive behavior is produced by housing mice in individual cages and
then introducing a stranger mouse. Anxiolytics reduce this aggression.

   Analgesics:
  - Narcotic analgesics e.g. morphine, methadone
  - Non-narcotic analgesics which are also used as antipyretic and
    anti-inflammatory drugs e.g. Salicylates, diclofenac

   Screening tests for narcotic analgesics

  I-    Mechanical methods:

  a) Tail clip method
The mouse tail (as well as the ear) is not covered by hair so it is very
sensitive to pain due to the presence of sensory nerves in the skin.

   An artery clip (clamp) is applied to the base of the mouse tail for 30
     seconds. The animal will feel pain and will try to get rid of the clip.
   A group of mice is treated with the test drug, and the clip is applied to
     the tail after 30, 60, 120 and 180 minutes. If the animal’s response to
     the clip is abolished or delayed, then the drug may have analgesic
     properties. The ED50 is calculated.

   b) Tail compression method
This test uses an instrument (analgesiometer) producing a gradual
increasing pressure on the tail which can be measured.
    The pressure is gradually increased till a certain threshold pressure
      (e.g. 20 mm Hg) is reached at which the animal squeaks.
    The mean threshold pressure in control
      animals is determined.
    The drug under test is injected I.P. and
      the mean        threshold pressure       is
      determined after 30, 60 120 and 180
      minutes. If the threshold pressure
      increases by 2 folds (e.g. 40 mm Hg) then
      the drug may be an analgesic.

  II-   Thermal methods:

  a) Hot plate method
   A mouse is put in a glass cylinder kept on a hot plate at 55 C and the
     reaction time is calculated (time from insertion of the animal inside
     the cylinder until it licks its feet or tries to jump out of the cylinder).
   The mean reaction time in a control group is usually < 1 minute.
   The drug under test (X) is injected into another group of mice and the
     reaction time is determined after 10, 30, 60, 90, 120 and 180 minutes.
     If the reaction time is increased by 3 folds (3 minutes for e.g.), the
     drug is considered as an analgesic.

  b) Tail flick method
   The rat’s tail is used, and an electric current is allowed to pass
     through a coil until it becomes bright red.

    The reaction time, which is the time elapsed between closing of
     electric circuit and withdrawal of tail, is recorded.

    Screening tests for non-narcotic analgesics
   I-   Chemical methods

Writhing method
In this method, moderate pain is induced in mice by chemicals like acetic
acid (300 mg/kg) or phenylquinone (0.2 ml of 0.02% solution) producing
seizures of abdominal cramps (i.e. cramps in the abdominal skeletal
muscles) after 15 min (positive writhing response).
 Acetic acid or phenylquinone are injected in a group of animals and
    ONLY the animals showing a positive response are selected.
 Next day, the same animals are injected with drug (X) then 1 hour later,
    with acetic acid. If no writhing occurs, the drug may be an analgesic.
 It is a quantal assay. Plot the relation between log the dose and the
    percentage protection. The ED50 for T and S are compared.

   II-   Electrical methods

  a) Pododolorimeter method
 A cage with a floor made of a series of metallic rods which can be
  electrified is used.
 Record the minimum volt, which causes the rat or mouse to cry or
  struggle in the absence and presence of analgesics.

  b) Tooth pulp method
 This method depends on electrical stimulation of a metal filling in the
  teeth of a dog.
 Observe the chewing movements or slight tremors of the jaw and rapid
  upwards thrust of the head and measure the volt in the absence and
  presence of analgesic drugs.

   III- Determination of antipyretic activity
Rectal temperature of the rats is recorded. The rats are then made febrile
by S.C. injection of yeast in a suspension of 15% gum Arabic (1 ml/100g
body weight). Five hours after yeast injection, the drug is given orally or I.P.
The rectal temperature is taken at almost hourly intervals for 7 hours.

    Screening tests for anti-inflammatory drugs
Anti-inflammatory drugs are used in the treatment of inflammation in many
diseases e.g. salicylates, cortisone derivatives.

   a) Formalin or carrageenan-induced edema in the rat hind paw
Formalin & carrageenan are inflammatory materials used to induce
experimental inflammation due to the release of inflammatory mediators
(histamine, kinins and prostaglandins).

 Carrageenan sodium gel (5 l of 1% solution) is injected S.C. in the rat
  hind paw causing inflammation and edema after 1 hour.
 The volume of edema in the hind paw is measured by volume
  displacement using mercury plethysmography. The hind paw is placed
  in mercury, causing mercury displacement. The displaced volume is
  measured by mercury manometer.
 Another group of rats is injected I.P. by the anti-inflammatory drug then,
  after 1 hour, carrageenan or formalin are injected into the hind paw.
  Anti-inflammatory drugs reduce the volume of edema and displaced

   b) Granuloma (cotton pellet) method
A foreign body like sterile cotton pellets, 5 mg each, are implanted in the 4
axillary and groin positions in the rat under ether anesthesia. The pellets
will be considered as foreign bodies leading to an inflammatory reaction.
After 1 week, the cotton pellets become surrounded by fibrous tissues
(granuloma), they are isolated from the animal and weighed (pellet +

 Pre-weighed cotton pellets are implanted in the 4 axillary and groin
  positions in control rats.
 In another group of rats, cotton pellets are implanted and the rats are
  injected daily with the drug under test for the following one week.
 After 1 week, rats are killed and the granuloma is obtained and
  weighed. Anti-inflammatory drugs decrease the weight of granuloma.

  c) Erythema method
 Inflammation (erythema) is induced in the shaved back of guinea pigs,
  through exposure to UV light for 20 seconds. Two hours later, the
  degree of erythema is graded on a scale from 0 to 4.
 In another group of guinea pigs, the drug under test is administered then,
  30 minutes later, animals are exposed to UV light for 20 seconds. The
  erythema score is determined after 2 hours.
 Erythema is due to release of serotonin; anti-serotonin drugs give
  positive test although they are devoid of anti-inflammatory effect.

   Antidepressants;
These drugs are used in the treatment of psychic depression e.g.
imipramine, fluoxetine

    Biological evaluation of antidepressants:
In vitro assays (Inhibition of monoamine uptake)
1. Inhibition of 3H-norepinephrine uptake in rat brain (hypothalamus)
2. Inhibition of 3H-dopamine uptake in rat striatal synaptosomes.
3. Inhibition of 3H-5-hydroxytryptamine uptake in rat striatal synaptosomes.

In vivo assavs
   a) Catalepsy antagonism in chicken.
The animal is grasped by hands and held for one minute.
Cataleptic numbness occurs immediately and is maintained
for a period of time. The cataleptic rigor is interrupted by any
noise or fast movement of the observer.
Test drugs (antidepressants) are considered to be positive if
the cataleptic rigor does not occur after treatment.

  b) Despair swim test (hopelessness model).
Mice or rats are forced to swim in a restricted space from which they
cannot escape. The animals develop a characteristic behavior of
immobility, which indicates a state of despair similar to depression in
humans. This state of immobility can be reduced by antidepressants.

   c) Tail suspension test in mice (behavioral despair)
Antidepressants reduce the immobility that mice display
after active and unsuccessful attempts when suspended by
the tail.
   d) Antagonism of behavioral effects of tetrabenazine.
Tetrabenazine depletes biogenic amines and causes ptosis (eye-lid
closure) and catalepsy in mice. These effects are prevented by

   e) Antagonism of reserpine-induced hypothermia.
Reserpine depletes biogenic amines in brain and causes hypothermia and
ptosis. These effects can be prevented by antidepressants.

   Antiepileptics:
Example of antiepileptic drugs are phenytoin and valproate

   Screening tests for antiepileptics
Methods of induction of experimental epilepsy include:
  I-   Electrical methods (preferred for grand mal epilepsy)

  a) Supramaximal electric shock
 Male mice are subjected to an electric shock at high voltage (140 V), for
  short duration (0.3millisecond) and at a frequency of 100 Hz. This will
  produce tonic and clonic convulsions.
 Animals are given the antiepileptic drug, the electric shock is repeated
  and the PD50 is determined (the dose that protects 50% of the animals).

  b) Psychomotor electric shock
 An electric shock of lower frequency (6Hz) at 50-100 volts for
  6 seconds is used. This will produce severe clonic seizures with loss of
  righting reflex for 10 seconds or the mice may become stationary for 10
  seconds before returning to normal position.
 The antiepileptic drug is given, the psychomotor electroshock is
  repeated and the PD50 is determined.
 Protection is considered when the animal becomes able to walk away
  within 10 seconds after shock delivery.

  II-    Chemical methods (preferred for petit mal epilepsy)

 Epilepsy in induced by chemicals such as Cardiazol® or Leptazol® (100
  mg/kg) injected I.P. in mice or rats or 3 mercaptopropionic acid (0.5 ml
  of 1.6% aqueous solution) in mice.

 Animals are injected with the chemical (tremors and convulsions
  indicate a positive test).
 Only animals showing the positive response are injected with the drug
  under test and then injected with Leptazol. The PD50 is determined.
 The threshold of the stimulus required to induce convulsions may be
  reduced by I.P. injection of 5.5% glucose which increases sodium
  excretion leading to hyponatremia that favors convulsions.

   Local anesthetics:
Example of these drugs are lignocaine & benzocaine

   Screening and bioassay of local anesthetics
  a) Frog plexus method
 The frog is decapitated and eviscerated leaving an
  abdominal pouch where the sciatic plexus is vulnerable.
 An irritant substance, e.g. dilute HCl, is applied to the leg
  for 10 seconds and the leg withdrawal reflex is observed.
 The onset time of the withdrawal response, (the time
  between HCl application and leg withdrawal), is
  plotted against log concentrations of the local anesthetic.

  b) Corneal anesthesia method
 The eye lashes of a guinea pig are removed and the cornea is
  stimulated by approaching a horse hair to the side of the cornea for 5
  minutes and the eye blink is observed. The cornea is stimulated again
  after instillation of the local anesthetic in both eyes.
 Determine the percentage failure of eye blink in a total 10 stimuli for
  each local anesthetic concentration and determine the EC 50.

   Drugs for Alzheimer’s disease:
These include drugs such as rivastigmine & physostigmine (central
cholinesterase inhibitors) or bethanechol, arecoline (cholinergic agonists)

    Biological evaluation of AD drugs

   a) Reversal of scopolamine-induced cognitive deficits
Scopolamine induces sedation, transient memory impairment and learning
deficits in normal subjects (a model of AD). These deficits are reversed by
I.M. physostigmine due to facilitation of central cholinergic transmission.

   b) Effects on sleep architecture
Cholinergic drugs such as arecoline and others lead to induction of REM
sleep (central cholinomimetic effect) due to M2 receptor stimulation
whereas Ml subtype is important for memory (disadvantage of the assay).

  c) Measurement of RBCs cholinesterase inhibition

   Antiparkinsonian drugs:
They include dopaminergic drugs e.g. L-dopa, or central anticholinergics
e.g. benztropine.

   Biological evaluation of antiparkinsonian drugs

  a) Tremorine and oxotremorine antagonism
Muscarinic agonists such as tremorine and oxotremorine induce
parkinsonism-like signs which are antagonized by anticholinergic drugs.

   b) MPTP model in monkeys
MPTP (methylphenyltetrahydropyridine) when administered to primates
causes partial destruction of basal ganglia causing a syndrome that
resembles Parkinson’s disease (akinesia, rigidity, tremors). These
symptoms may be reversed by the administration of L-dopa.

   c) Reserpine antagonism
Reserpine induces depletion of central catecholamine stores. The sedative
effect can be observed in mice shortly after injection, followed by signs of
eyelid ptosis, hypokinesia, rigidity and immobility. These phenomena can
be antagonized by dopamine agonists.


  1- What is the difference between conditioned and unconditioned
     avoidance responses?
  2- What is the specific screening test for antipsychotics?
  3- What is meant by:
         Sleeping time
         Positive writhing response
  4- Discuss the methods of experimental epilepsy that are preferred for
     grand mal epilepsy.


     How can psychotropic drugs be assayed by causing changes in the
     animal behavior?

Student ID: from 106121 to 106150

Presentation format:
Word document, not more than 2 pages, including student data (name, ID)
font type: Arial, font size: 14, line spacing: 1.5, margins: 3 cm around.


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