PHARMACOLOGICAL & TOXICOLOGICAL

SCREENING METHODS-I
(PTSM-I)

Dr.R.Vadivelan
Professor in Pharmacology

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Unit-I
Laboratory Animals
 Common laboratory animals: Description, handling and
applications of different species and strains of animals.
 Transgenic animals: Production, maintenance and
applications
 Anaesthesia and euthanasia of experimental animals.
 Maintenance and breeding of laboratory animals.
 CPCSEA guidelines to conduct experiments on animals
 Good laboratory practice.
 Bioassay-Principle, scope and limitations and methods

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BIOASSAYS
 Topics to discuss

 Introduction
 Indications of bioassay
 Applications of bioassay
 Principles of bioassay
 Types of bioassay
 Criteria for good biological assay
 Dose response curve
 Some basic instruments used for isolated tissue
experiments
 PSS

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 Introduction

◦ Estimation of the conc / potency of a substance by

measuring its biological response in living systems
◦ i.e. Observation of pharmacological effects on
 Living tissues, or cells
 Microorganisms
 Animals

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 Indications for Bioassay
 Active principle of drug is unknown
 Active principle cannot be isolated, e.g. insulin, posterior pituitary
extract etc.
 Chemical method is either
◦ not available
◦ if available, too complex,
◦ insensitive to low doses e.g. Histamine can be bioassayed in microgram
conc.
◦ When the bioassay is more sensitive than the chemical assay
 Unknown chemical composition but the substance has a specific
biological action, e.g. long acting thyroid stimulator.
 Chemical composition of drug variable but has same pharmacological
action e.g. cardiac glycosides isolated from diff sources, catecholamines
etc.

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 Principles of Bioassay

1. All bioassays must be comparative against a standard drug or

preparation (Reference preparation-India CDRI-Calcutta)
2. The standard and the new drug should be, as far as possible
identical to each other.(DRC is parallel)
3. The method for comparing the unknown and the standard drug
should preferably (but not essentially) test the therapeutic
property of the drug (Ideally an analgesic should be tested for
analgesic activity)
4. The method should estimate as far as possible and allow an
estimate of the error due to biological variation in
different animals/ persons at any time and in the same
animal/person at different times

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 Precautions to be taken to minimize
the errors due to biological variations
 The experimental conditions must be kept constant
 Biological response(indicator) should be sensitive to the drug
 Indicator should be insensitive to other drugs
 Indicator should capable of giving constant and reproducible results
 A preparation of known concentration must be available to
compare the response
 No. of experiments should be sufficiently large and the assay
designed to minimize the biological variation
 Animals should be of same strain and species, similar age, sex and
weight, kept under similar diet and housed under similar
conditions
 In certain bioassays designs cross-over of animals receiving the
sample and the standard is possible

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 Types of Bioassays
 Quantal assays [ Direct endpoint ]
◦ Elicits an ‘All or None’ response
◦ Examples
 Digitalis induced cardiac arrest in guinea pigs
 Insulin induced hypoglycemic convulsions in mice.
 t-tubocurarine induced head drop in rabbits
 Calculation of LD50 in mice or rats
 Graded response assays [mostly on tissues]
◦ Graded responses to varying doses
◦ Unknown dose response measured on same tissue

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 Methods of Bioassay contd..
 Graded Response Assays
[ Direct comparison on same tissues]
◦ Interpolation: Conc. of unknown is read from a standard plot
of a log dose response curve of at least 4 sub maximal
concentrations
◦ Matching / Bracketing: Const dose bracketed with varying
doses of standard till exact match is obtained
 Used when test sample is too small
 Inaccurate & margin of error difficult to estimate
 Eg. Histamine on guinea pig ileum, oxytocin on rat uterus
◦ Multiple Point Assays
 3 point assay [combines pples of matching with interpolation]
 4 point assay [combines pples of matching with interpolation]

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 3 point assay [2+1 dose assay]
 Fast & convenient
 Procedure [eg. Ach bioassay]
 Log dose response curve plotted with varying
conc. of std Ach solutions and given test solution
 Select two std doses S1& S2 [ in 1:2 dose ratio]
from linear part of LDR [ Let the corresponding
response be S1, S2]
 Choose a test dose t with a response T between
S1 & S2
 Record 4 sets data [Latin square: Randomisation
reduces error] as follows
 S1 S2 t
 S2 t S1
 S1 S2 t
 Plot mean of S1, S2 and T against dose. Calculate
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3 point assay [2+1 dose assay]

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 4 point assay [2 +2 dose assay]
 Procedure [eg. Ach bioassay] Log dose response [LDR] curve
plotted with varying conc of std Ach solutions and given test
solution
◦ Select two std doses S1& S2 from linear part of LDR
[ Let the corresponding response be S1, S2]
◦ Choose two test doses T1 & T2 with response T1 &T2
between S1 & S2 ; Also S2/S1 = T2/T1 = 2
◦ Record 4 data sets [Latin square: Randomisation
reduces error]
 S1 S2 T1 T2
 S2 T1 T2 S1
 T1 T2 S1 S2
 T2 S1 S2 T1
◦ Plot mean of S1, S2 and T1, T2 against dose. Calculate

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Some basic instruments used for isolated tissue
experiments

Fig. 1.Student’s Organ Bath—A : Outer jacket; B : Organ bath; C : Thermostat;

D : Stirrer; E :Oxygen tube; and F : Glass coil.

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 Recording levers
 They are used to record the contractions or relaxations of the
isolated tissue preparation.
 The recording is done on smoked papers fixed on circular cylinders
(of different diameters) and run at different speed using electrical
recording drums.
 The speed of the drum is adjusted depending upon the nature of
the experiment.(0.12mm/sec)

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 Drum speed=0.12mm/sec

Sherrington Rotating Drum

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 Recording levers
 The writing levers are light in weight, rigid and are generally made
up of wood (straw), light aluminium or stainless steel The levers are
of two types—
 (1) Isotonic type, i.e. change in length due to contraction is
recoiled while the tension on the muscle remains the same. The
examples of isotonic levers are simple lever, frontal writing lever,
and
 (2) Isometric type, i.e. isometric recording measures increases in
tension of the tissue when the length of the tissue is kept constant.
These are used in special circumstances such as recording muscle
twitches produced by electrical stimulation. For recording such
observations isometric strain-gauge transducer may be preferred

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 Common recording levers

Simple lever (side-way writing)—It is the simplest type of lever made up of

wood, stainless steel or aluminium. A celluloid writing tip (stylus) is attached at
the end of the longer arm. The contractions are recorded as curved lines.
Frontal writing lever (writes frontally)—This lever is designed in such a way
that the writing point rotates freely about its axle. This helps in reducing the
tension between the smoked paper and the recording tip. The contractions are
recorded as straight line.
Starling's heart lever—This lever is used to record the contraction of the heart.
The difference between this and other isotonic levers is that the fulcrum lies at
one end beyond the point of attachment.
Brodie's universal lever—-It is a general utility lever.
The other levers and essential equipment used along with organ bath are
gimbal lever, auxotonic lever, straw or lever holder (fulcrum), different types of
X-blocks, clamps, supporting rods, thermometer and surgical instruments.

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C

Fig. 1.4 Recording Levers

A: Simple lever; B: Frontal writing lever,
C: Starling's heart lever; D : Brodie's lever

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 Adjustment for magnification

 Depending on the inherent contractility of the tissue preparation

under study, the magnification of the response should be adjusted
in order to get a proper recording of the observed physiological
response.
 The tissues showing less contractility need more magnification and
the reverse is true with tissues which have higher inherent
rhythmic contractility. For example, while recording the effects on
guinea pig ileum or rectus abdominis muscle it is desirable
to have 5-10 fold magnification whereas, for rat uterus
preparation the magnification needed is only 4-6 times.
 The adjustment for magnification is done by properly adjusting the
distance between the writing tip and the fulcrum, and the distance
between the point of attachment to the tissue and the fulcrum. By
adjusting relative distances desired degree of magnification is
obtained

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 Adjustment for magnification

Distance between fulcrum and writing point (A)

--------------------------------------------------------------
Magnification value = Distance between fulcrum and the point of attachment to the tissue (B)

If the distance of the longer arm (A) is 10 cm and that of the shorter arm (B) is 2
cm, the magnification (A/B) will be 5.

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 Application of load {tension)
 The muscle preparation has to be properly relaxed without affecting the
normal tone and rhythmic activity so that efficient contractions are achieved
when stimulated, and it also relaxes to its full length afterwards.
 This is achieved in the following way—
 (i) select the proper length of the longer and shorter arms depending on the magnification for
the tissue which is under study, and fix the fulcrum;
 (ii) balance the lever by putting the weight (plasticine) at the end of the shorter arm and
mark the point of tissue attachment;
 (iii) at equidistance, i.e. the distance between the fulcrum and the point of tissue attachment,
from the fulcrum on the longer arm of the lever, fix the desired load (plasticine) required for
the particular tissue.
 The tension (load) prescribed for various commonly used tissue preparations
are—guinea pig ileum (1 g); guinea pig trachea (0.2 g); guinea pig vas deferens
(0.5 g); rabbit duodenum (1-3 g); rat uterus (1 g); rat colon (0.5 g); rat fundus
(1 g) and frog rectus abdominis (1-5 g), respectively.

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 Contact time
 The time that is allowed for the drug (agonist) to remain in contact
with the tissue is called the contact time. The contact time depends
upon the type of the tissue used.
 For example, a slow contracting tissue such as frog rectus
abdominis preparation, the contact time allowed is 90 sec.
 On the other hand, for guinea pig ileum the contact time is 30 sec.
 Rat colon-60 secs
 When the drug is in the vicinity of receptors it is called in
'biophase' or 'receptor compartment'

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 Time cycle
 A fixed time cycle is used while recording any effect of the drug on
the isolated tissue preparation.
 The fixed time cycle which comprises of starting of the drum,
recording the base-line, effect of the drug (contact-time) and
washout period.
 Generally a five minute time cycle is followed i.e. 30 sec. of the base
line recording, 90 sec. of contact time (response of the drug) and
the subsequent three washings at an interval of each minute.
 It is very essential to follow the fixed time cycle while doing the
bioassays to obtain uniform recordings and, it also helps in
calculating the approximate time required to complete the
experiments depending upon the number of effects to be recorded

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 PHYSIOLOGICAL SALT SOLUTION (PSS):
 The ionic requirements and nutritional supply can be provided by
using the suitable solution, commonly known as physiological salt
solution.
 Its composition is such that it provides an artificial media
resembling the inorganic composition of blood plasma together
with a buffer mechanism to maintain the optimum pH about 7.0 to
7.2 and glucose to facilitate tissue metabolism.
 Commonly used PSS are
◦ Frog Ringer (for frog heart, rectus abdominis )
◦ Tyrode (for guinea pig ileum, rat ileum, rabbit ileum etc.),
◦ de Jalone (for rat uterus),
◦ Krebs’s solution (for rat fundus strip, tracheal preparations,Vas
deferens) etc

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 COMPOSITION OF PHYSIOLOGICAL SALT SOLUTION (PSS)

Compound Frog Ringer Ringer or De Jalon Tyrode Kerbs

ringer locke Hensleit
(Locke) (Krebs)
Nacl (58.45)* 110(6.0)** 154(9.0) 154(9.0) 137(8.0) 118(6.9)
Kcl (74.56) 1.9(0.14) 5.6(0.42) 5.6(0.42) 2.7(0.2) 4.7(0.35)
Cacl2(110.99) 1.1(0.12) 2.2(0.24) 0.55(0.06) 1.8(0.2) 2.5(0.28)
Mgcl2(95.23) _ _ _ 0.1-1.0 _
(0.01-0.10)
MgSo4.7H20 _ _ _ _ 1.2(0.16)
NaHCO3 2.4(0.2) 6.0(0.5) 6.0(0.5) 11.9(1.0) 25.0(2.1)
NaH2PO4(119.97) _ _ _ 0.4(0.05) _
KH2PO4(136.08) _ _ _ _ 1.2(0.16)
Glucose(180.16) 11.1(2.0) 5.55(1.0) 2.78(0.5) 5.55(1.0)or 5.55(1.0)or
11.1(2.0) 11.1(2.0)

*Values in parenthesis against salts indicate mol.wt.

** Values are in mM(g/l)

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 Purpose of each ingredient

 Sodium Chloride (Nacl): To maintain iso-osmolarity, isotonicity,

excitability and contractility of the preparation.
 Potassium Chloride (Kcl): To maintain the ionic balance
 Calcium Chloride (CaCl2): To maintain the contractility of
the preparation
 Sodium bicarbonate (NaHCO3): To provide alkaline pH
 Glucose: To provide energy
 Sodium or potassium dihydrogen phosphate (NaH2PO4 or
KH2PO4): Acts as a buffer.
 Magnesium chloride or sulphate: To stabilize the preparation
and hence to reduce the spontaneous activity

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