Bio-Assay Pharmacology
&
Dose Response Relationship
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BIOASSAY
Definition: Assay of the drug or chemical by using Biological System.
Biological System: In-Vitro (living Tissues, cells, microbes) & In-Vivo
(Animals)
Types
1. Qualitative Bioassay: Qualitative estimation of biological response or
pharmacological effect of a drug.
✓ Examination of response +ve or –ve only
✓ Not related to dose/concentration of a drug or chemical
✓ E.g. Behavioral effects, Analgesic effects, Contraction or Relaxation
BIOASSAY
2. Quantitative Bioassay (Quantitative estimation): Estimation of the
concentration / potency of a drug/ chemical by measuring its
biological response in living systems
✓ Examination of biological response in respect to dose or conc.
✓ It is related to dose/concentration of a drug or chemical
E.g. Dose estimation of a toxic agent for
toxic response.
NOAEL: No observed adverse effect level
LOAEL: Lowest observed adverse effect
level
Importance/ Reason of Bio-Assay
❑ Chemical Assay is either:
✓ not available
✓ if available, too complex,
✓ insensitive to low doses e.g. Histamine can be bioassayed in microgram
conc.
❑ Active principle of drug is unknown and cannot be isolated, e.g.
insulin, posterior pituitary extract etc.
❑ Unknown Chemical composition, 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.
Application of Bioassay
Used in determination of drugs potency
Screenings of new agents isolated from plants, animals or chemical labs and
find their field of activities
Establishment of SAR (structure activity relationship)
Essential in monitoring environmental pollutants
Determination of the pharmacological activities of a new drug
Determine the therapeutic advantage of one drug over another treatments
Useful in study of new hormonal or other chemically mediated control
systems
Principles of Bio-Assay
❑ The basic principle of bioassay is to compare the test substance with
the International Standard preparation of the same and to find out
how much test substance is required to produce the same biological
effect, as produced by the standard.
Potency: concentration or dose of
drug, in which it produces 50%
response (EC50 and IC50)
Potency: A>B>C>D
Efficacy: A=B=C=D
Principles of Bio-Assay
Efficacy: concentration or dose of drug, in which it produces 100%
response (maximum/ceiling Effects)
Efficacy: A>B>C>D
Bioassay Methods
1. Quantal Bioassay (Direct Method): End Point Bioassay, All or None’ response
in different animals. E.g.,
Digitalis induced cardiac arrest in guinea pigs
Insulin induced Hypoglycemic convulsions in mice.
Calculation of LD50 in mice or rats
2. Graded Assay: Response to varying dose
Matching assay
Bracketing assay
Interpolation assay
Multiple point assay (3 point, 4 point, 6 point bioassay)
Quantal Bioassay
Digitalis Induced Cardiac Arrest:
The extract containing digitalis (standard or unknown) infused into vein of cats till
the heart stops to beat. Volume of fluid passed into the vein noted (threshold
dose).
Potency is calculated as follows:
Conc of Unknown = (Threshold dose of std/ Threshold dose of test) x Conc of std
Advantages of Quantal Bioassay:
Drug effects appears rapidly and easily recognized.
Effect directly proportional to drug dose.
Rapid end point detection.
Disadvantage of Quantal Bioassay:
Only toxicity or high dose study possible.
Dose ranging study can‘t be done.
Graded Bioassay
An assay may be based on a graded response with dose
e.g. change in blood glucose concentration, contraction of a strip of
smooth muscle, change in the time taken for a rat to run a maze.
Figure. Dose response curve (DRC) of histamine on ileum
Matching Bioassay
❑ Test response is matched with the standard on same tissue
e.g. Std dose (mcg) - 16 mcg, Test dose (ml) = 0.2 ml
Conc. of unknown= Std dose (mcg)/ Test dose (ml)
16/0.2 = 80 mcg/ml
Bracketing Bioassay
Test drug response is bracketed between lower and higher response of
standard. S1 (30 ug), T(0.2 ml), S2 (50ug)
Bracketing assay of ACh on Frog Rectus Abdominus muscle
Interpolation Bioassay
Conc. of unknown is read from a standard plot of a log dose response curve
of at least 2-3 sub maximal concentrations
Multiple Point Bioassay
Three Point Bioassay : 2 std + 1 Test
DRC of std is plotted. Select two std doses s1& s2 [ in 1:2 dose ratio] from linear part
of DRC
Choose a test dose t with a response T between S1 & S2
Record 3 sets data as follows:
Unknown conc. Can determine by:
1. Graphical : Plot mean of S1, S2 and T against dose.
Multiple Point Bioassay
Mathematically:
Where, n1 = Lower Standard dose
n2 = Higher Standard dose
t = Test dose
S1 = Response of n1
S2 = Response of n2
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