PHARMACOLOGICAL AND TOXICOLOGICAL

SCREENING METHODS I(MPL 103T)

 BIOASSAY

Presented to
Mrs. MANISHA KAWADKAR Mem (Associate Professor)
 OVERVIEW

 BIOASSAY – Definition/Synonyms
 PRINCIPLES OF BIOASSAY
 INDICATIONS OF BIOASSAY
 TYPES OF BIOASSAY
 USES OF BIOASSAY
 DRAWBACKS
 BIOASSAY IN HUMANS
 W H AT I S A S S AY

Physicochemical assay
Biological (Bioassay)

o Types:

Immunological assay
 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

 SYNONYMS

Biological assay
Bio metrics

Biological Biostandadization
standadization
 P R I N C I P L E S O F B I O A S S AY

• 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

• 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

 I N D I C AT I O N S O F B I O A S S AY

 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..
 I N D I C AT I O N S O F B I O A S S AY

 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

 C H A R A C T E R I S T I C S O F A G O O D A S S AY
METHOD

• Sensitivity
• Specificity
• Repeatability
• Reproducibility
• Precision
• Accuracy
• Stability – tissue has to stay “bioassay-fit
 B I O A S S AY C A N B E P E R F O R M E D O N

Invivo • Intact animals

• Isolated tissues
Invitro • Specific cells
• Organisms
• 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 cells14

 T Y P E S O F B I O A S S AY

 QUANTAL ASSAY  INDIRECT ASSAY

 GRADED ASSAY  DIRECT ASSAY
 Q U A N TA L A S S AY

 Quantal response - the response is in the form of "all or none", i.e. either no response or
maximum response

 Drugs producing quantal effect can be bioassayed by end point method

 The threshold dose producing a predetermined effect is measured

 Comparison between the results of standard and the test

 E.g: Bioassay of digitalis in cats,Insulin induced hypoglycemic convulsions in rat

• Conc of Unknown = Threshold dose of the Std X Conc of Std

Threshold dose of the Test
 G R A D E D A S S AY

 Graded response - response is proportional to the dose and response may lie between no
response and the maximum response.
 Types:
• Bracketing /direct matching
• Interpolation
• Multiple point assays
I. Three point assay
II. Four point assay
III. Six point assay
• Cumulative dose response
standard Test/unknown
1
• DRC & Log DRC
 30%
 70%
 Sigmoid curve
 Wide range of doses can plot
 Rectangular hyperbola
 • Potency
 • Efficacy
 • Slope of curve
 B R A C K E T I N G O R D I R E C T M AT C H I N G

o A constant dose of the standard is bracketed by varying dose of test sample

o until an exact matching between the response of std & that of the sample is achieved

o Strength of unknowm/test drug can be found by simple interpolation of bracketed response.

B R A C K E T I N G O R D I R E C T M AT C H I N G
 A D VA N TA G E S

• Simple & Faster

• Amount of test drug available is small

• Does not involve complicated calculations

• Does not depend on DRC

 D I S A D VA N TA G E S

• less accurate,time consuming, troublesome

• cannot get exact match of response

• quantitative difference b/w test & std not obtained

 I N T E R P O L AT I O N A S S AY

• A log dose-response curve is plotted with the standard on a simple graph paper
or Semi-log paper

• The concentration of the test is then read from the graph

standard Test/unknown
 A D VA N TA G E S

• Sensitivity of tissue is 1st determined by prior plotting of a conc-response

curve with known agonist

• Dose can be plotted even if it varies over thousand fold range

• Error is normally distributed

 D I S A D VA N TA G E S

• Sensitivity of tissue changes with time

• Timing of doses not taken into account

• Variation in mode of application of drugs

 M U LT I P L E P O I N T A S S AY S

 Responses are repeated several times and the mean of each is taken
 Chances of error are minimized
 3 point method - 2 doses of std+1 dose of test
 4 point method - 2 doses of std+2 doses of test
 6 point method - 3 doses of std+3 doses of test
 Latin square
3 - P O I N T A S S AY

t s1 s2
s1 s2 t
s2 t s1
3 cycles
 L AT I N S Q U A R E D E S I G N

• s1 s2 t
• t s1 s2
• s2 s1 t
• t s2 s1
 C A L C U L AT I O N

• CALCULATION

• Mean responses of these 3 sets plotted

• Log potency ratio (M) = (T-S1÷ S2-S1)× log d

where, d – dose ratio = s2/s1

• Strength of unknown = s1/t × antilog of M

4 - P O I N T A S S AY
 L AT I N S Q U A R E D E S I G N

• s2 t1 t2 s1
• t1 t2 s1 s2
• t2 s1 s2 t1
• s1 s2 t1 t2
 C A L C U L AT I O N

• Mean responses of 4 sets plotted

• Log potency ratio (M)

• (T2-S2)+(T1-S1) × Log d
(S2-S1)+(T2-T1)

where, d-dose ratio = s2/s1

• Strength of unknown = s1/t1 × antilog of M

 S I X P O I N T A S S AY

 3+3 dose assay

 3 conc each of std & test drug are used
 6 sets of experiments using 6 doses in each set
 More time consuming,lesser in use
 Reliability is excellent
 C U M U L AT I V E D O S E R E S P O N S E C U RV E

• Increase conc of drug in bath fluid step by step without washing out the
preceeding doses

• Continue till supramaximal effect is seen

• Dose response curve is plotted

C U M U L AT I V E D O S E R E S P O N S E
 U S E S O F B I O A S S AY

• 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
 U S E S O F B I O A S S AY

• Determination of potency, ED50/LD50 of drugs

• New drug development

• Measure clinical effectiveness

 D R AW B A C K S

 Biological variation

 Troublesome

 Time consuming

 Expensive

 Less accurate than physico-chemical methods

 H U M A N T I S S U E B I O A S S AY

• Veins [surgery on varicose veins]

• Tissues like larger blood vessels obtained during amputation

• Organs removed during transplantation/ tumor surgeries/procedures requiring

resection

• Tissues collected Postmortem

 CONCLUSION

 Successful tool in estimation & discovery of biologically active substances

 Sensitivity & Specificity – important tool in pharmacology