Brief introduction on common laboratory animals and Bioassay

1. EXPERIMENTAL
PHARMACOLOGY
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2. COMMON LABORATORY
ANIMALS
The common laboratory animals used are
classified as follows:
 Rodents
Eg:- Mouse, Rat, Guinea Pig, Gerbil, Hamster, etc.
 Non-Rodents
Eg:- Rabbit, Dog, Cat, Monkey, etc.
 Miscellaneous
Eg: - Frog, Pigeon, Zebra Fish, Chicken, etc.
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3. The Rat (Rattus norvegicus)
Albino rat is one of the commonest laboratory
animals suitable for experimental work because
of its small size and greater sensitivity to most
drugs. It is also the most standardized of all
laboratory animals.
There are two original strains of albino rats used
are:
 Wistar Rats: Wide head and ear is long where as
tail length is less than body length.
 Sprague-Dawley Rats: Longer and narrow head,
tail is longer than the body length.
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4. The Guinea Pig (Cavia
porcellus)
 Guinea pig has proved to be the most useful
laboratory animal.
 Like other rodents, it is easy to keep them in
captivity.
 They differ from other rodents, being docile in
nature and very susceptible to tuberculosis
and anaphylactic shock.
 They are highly sensitive to histamine.
 Guinea pig are not able to synthesize the
daily required Vit-C.
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5. The Mouse (Mus musculus)
 Smallest laboratory animals
 Easy to keep, handle and require small
place for housing and uniformly breed.
 Swiss albino mouse is the most widely
used strain for laboratory investigations.
 Sensitive to small doses of drugs.
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6. The Hamster
 Third commonly used laboratory animal.
 Two species of Hamsters commonly used
are:
1. The Syrian or Golden Hamster
(Mesocricetus auratus)
2. The Chinese Hamster (Cricetulus griseus)
 Hamsters have chunky body with short
legs. They have prominent cheek pouches
extending up to the shoulder region. A
diminutive fluffy tail and rounded ears.
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7. The Rabbit (Oryctolagus
cuniculus)
 Rabbits are very docile animals employed
for variety of studies.
 New Zealand white rabbits are widely used
for the purpose.
 Used for testing large volume parentrals
and for the screening of Bioassay of
insulin, anti-diabetic and curare mimetic
drugs.
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8. The Cat (Felis catus)
 Used in cardio vascular behavioral and
biomedical research.
 Has distinct nictitating membrane, used in
screening of ganglionic drugs.
 Cats are useful models in studying the
transmission of vitamins and minerals to the
fetus and newborn.
 It is also used in neuropharmacology,
toxicology, oncology and chromosomal
abnormality studies.
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9. The Dog (Canis familiaris)
 Most preferred large experimental
animal used for studying of various
anti-arrhythmic, cardiovascular and
autonomic drugs.
 The advantages are small
alimentary tract and easily get
trained.
 Mongrel and Beagles are the most
preferred species.
Mongrel
Beagles
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10. The Monkey (Macaca mulatta)
 Structurally and functionally similar to
man.
 Suitable for undertaking psycho-
pharmacological studies.
 It is an ideal model for pharmacokinetic
study.
 Best for studying drugs acting on CNS,
CVS, GIT and Fertility.
 Used as Primate model to study drug
metabolism.
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11. The Frog (Rana tigrina)
 Commonly used amphibian in
pharmacology laboratory.
 Oxygen can pass through their highly
permeable skin and hence “breathe”
largely through their skin.
 Camouflage is a common defensive
mechanism in frogs.
 Anatomically their heart contains three
chambers which is different from the
other mammals used in the
experiments.
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12. Biological and Physiological
data of laboratory animals
Parameter Mouse Rat Guinea pig Rabbit
Typical adult wt. (gm) 20-40 250 800 1500-5000
Avg. life span (yrs) 1.5-2.5 2-3 3-5 4-6
Avg. age for
experiment
3 wks 6 wks 3 mnths 6 mnths
Gestation period (days) 21 (3 wks) 21 (3 wks) 68 (2
mnths)
31 (1 mnth)
Avg. Litter size 6-12 8-10 3-4 6-8
No. of Litters per year 8 6 4 4
Heart rate (bpm) 350-750 300-500 250-400 130-300
BP 113/81 116/90 77/47 110/80
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13. What is ASSAY?
 Amount or activity of an active principle in unit
quantity of preparation.
 Types:-
Physico-
chemical assay
Biological assay
(Bioassay)
Immunological
assay
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14. BIOASSAY
 Potency or concentration of an
active principle in unit quantity of
preparation.
 by measuring its biological
response on living tissues.
 Introduced by Paul Ehrlich -
biostandardization of Diphtheria
antitoxin.
Biological
Assay
Biometrics
Biological
Standardization
Bio-
standardizatio
n
Synonyms
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15. Principles of Bioassay
 To compare the test substance with the International Standard preparation of
the same.
 To find out how much test substance is required to produce the same
biological effect, as produced by the standard.
 Activity assayed should be the activity of interest.
 Standard & test sample - similar pharmacological effects & mode of action.
 Both should be compared for their established pharmacological effect using
specified technique.
Ex: *Ach – contractile response on frog rectus
*Histamine – contractile response on guinea pig ileum
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16. Cont….
 Problem of biological variation must be minimized.
 Experimental conditions - kept constant
 Animals - same species, sex and weight
 Number of animals - large enough to minimize error
(individual variation)
 Isolated preparations - sensitive
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17. Indications of Bioassay
 No chemical method has been developed
 Chemical assay is too complex /not sensitive enough to measure (ex: insulin, Ach)
 To measure the pharmacological activity of new or chemically undefined substances
 For biological standardization of drugs obtained from natural sources as these
cannot be obtained in pure form. Eg: Oxytocin, Vasopressin, Insulin, Heparin.
 To compare the strength of a drug obtained from various sources due to different
compositions (Eg: Cardiac glycosides)
 Chemicals with similar structure, but different biological activity.
 Chemical structure of the active principle is unknown.
 Chemical structure known; cannot be actively purified. Eg: Peptide hormones
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18. Characteristics
of a good assay
method:
• Intact
animalsIn
vivo
• Isolated
tissue
• Specific cells
• Organisms
In
vitro
 Sensitivity
 Specificity
 Repeatability
 Reproducibility
 Precision
 Accuracy
 Stability – tissue
has to stay
“bioassay-fit”
Bioassay can be performed on
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19. WHOLE ANIMALS
• Nor adrenaline – Spinal cat
• Cardiac glycosides – Guinea
pig
• Insulin – Mice
• Estrogens – Ovariectamised
female rat
MICRO ORGANISMS
• Vit B12 – Euglena gracilis
• Tetracycline - Bacillus pumilus
ISOLATED TISSUE
• Acetyl Choline – Frog Rectus
Abdominus muscle
• Histamine – Guinea Pig ileum
• Adrenaline – Rat uterus
• Oxytocin – Rat uterus oestrogen
primed
DISPERSED CELLS
• Plasma LH estimation by
stimulation of testosterone
synthesis - on isolated Leydig
cells
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20. Uses of Bioassay
 To measure the pharmacological activity of new/chemically undefined
substances.
 To investigate the function of endogenous mediators.
 To measure drug toxicity and unwanted effects.
 To measure the conc. Of drugs and other active substances in the
blood or other body fluids.
 Determination of potency, ED50/LD50 of drugs.
 New drug development
 Measure clinical effectiveness.
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21. Drawbacks
 Biological variation
 Troublesome
 Time consuming
 Expensive
 Less accurate than physico-chemical
methods
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22. Types of Bioassay
Qualitative Bioassay
 Is used for assessing the physical
effects of a substance that may not be
quantified, such as abnormal
development or deformity.
 Eg: Arnold Adolph Berthold's famous
experiment on castrated chickens. This
analysis found that by removing the
testes of a chicken, it would not develop
into a rooster because the endocrine
signals necessary for this process were
not available.
Quantitative Bioassay
 involve estimation of
concentration/potency of a
substance by measurement of
the biological response it
produces.
 These bioassays are typically
analyzed using the methods of
biostatistics
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23. Bioassay Classification
1
• Direct end point assay
(DEPA)
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• Quantal assay (All or
none assay)
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• Graded assay
a) Bracketing assay
b) Matching assay
c) Interpolation assay
d) Multiple point assay (3-
point, 4-point, 6-point, 8-
point)
Types
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