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Bioassay - Estimation of the conc./potency of a substance bymeasuring its biological response in living systems

Determination of potency of drug in biological tissue known as

bioassay

Where asBio standardization refer to determination of its limit

according to pharmacopoea

But in common practice, both term use interchangeably

It can be done either on Intact animal or isolated tissue

Bioassay of an agent performed on subject with great inherent

sensitivity to that agent

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Unknown drug always compared with reference standard

Standard should be highly purified, stable preparation

Its source, method of preparation, potency & use should

clearly defined Biological standard can be

Primary standard

Secondary standard

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Effect of drug can assess on living tissue, so give better idea

about drug action

It can be perform with very low dose

Screening of biological activity

To testing toxicity of newly developed chemical

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Comparison of pharmacological response of unknownpreparation with standard

Reference standard & test sample should have similar

pharmacological effect with same mode of action

Test solution & reference standard should be compared for their

established pharmacological effect

Method selected should be reliable, sensitive, reproducible &should minimize error due to biological variation &

methodology

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Measure pharmacological activity of new or chemicallyundefined substances

Measure the concentration of known substance in tissue or body

fluid

Measure effective dose(ED50) & lethal dose(LD50) of a drug

Bio standardisation of drugs from natural sources which

cannot be obtained in chemically pure form

e.g.- vasopressin, oxytocin

Investigation of the function of endogenous mediators

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Determination of the side-effect profile including the degree

of drug toxicity

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Quantal assay

Graded response assay

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Direct endpoint assay

Based on All orNone response

Criteria here is end point(i.e. response) , should be clear cut

Conc. at end point is referred as Threshold dose

E.g.- Head drop assay for d-tubocurarine in rabbits,

digitalis induced cardiac arrest in guinea pigs

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In toxicity studies, animal receiving a dose of drug either

dies or does not die

Quantal method employed for bioassay of substance in the

following ways:

Comparison of threshold response

Comparison of effective dose (ED50) or median lethal dose (LD50)

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Mostly on isolated tissues

Responses to varying doses of drug are

graded & measured

Effect of standard & unknown drug

measured repeatedly on same tissue

E.g. Ach on frog rectus abdominis , histamine on guinea

pig ileum

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Choice of procedure depends on:

Precision of assay required

Quantity of sample available

Availability of experimental animals

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Following method employed :

Matching assay

Bracketing assay

Interpolation method

Multiple point assay

3 point assay

4 point assay

6 point assay

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Simplest type of bioassay

Employed when sample size is very small

Dose response curve plotted with varying conc. of standard

& test solution Test response matched against response of reference

standard

Margin of error difficult to estimate Precision & reliability not very good by this method

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Dose response curve plotted with varying conc. of standard

& test solution

Dose of test is bracketed between 2 doses of reference

standard

Tissue should be very sensitive & have good

discriminating power

Particularly used when sensitivity is not steady for long

period Employed when sample size is small

Precision & reliability is poor

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Concentration-response curve of standard substance first

established

Record 2-3 responses from test substance

Selection of test such that they lie on linear portion of log

dose response curve of standard

Precision & reliability of this assay much better thanmatching assay

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Matching, bracketing, interpolation are not reliable

methods as they not take into account:

Changes in sensitivity of tissue with time

Variation in mode of application of drugs

So, error is more compare to multiple point assay

Hence, not subjected for statistical evaluation

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Fast & convenient; accuracy is lesser compare to four & sixpoint

Procedure:

Dose response curve (DRC) plotted with varyingconcentration of standard & test solution

Select 2 standard doses s1 & s2 with responses S1, S2 from

standard DRC (1:2 ratio)

Choose a test dose t with a response T between S1 & S2

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Record 4 sets from s1, s2, t in randomized manner

s1 s2 t

t s1 s2 s2 s1 t

t s2 s1Latin square design for 3 point assay

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Mean responses calculated from 4 sets & plotted on graphpaper

Calculate , Log Potency ratio [M]

M = [(T S1) / (S2-S1) ] X log dWhere d is dose ratio

From M, Strength of unknown can be calculated

Strength of test =s1 / t antilog of M

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Cumbersome , time consuming, provide fairly accurate result

Procedure:

DRC curve plotted with varying concentration of standard &

test solution Select 2 standard doses s1 & s2 with responses S1, S2 from

standard DRC

Choose 2 test doses t1 & t2 with response T1 &T2 such thatT1

lie between S1 & S2, T2 S2

Ratio of s1,s2 & t1,t2 should be 1:2

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Record 4 sets(using s1, s2, t1, t2) as per Latin square design

Mean responses calculated & plotted on graph paper

From these data, log potency ratio(M)

M = [ (T1 S1 + T2 S2) / (S2-S1 + T2-T1) ] X log d

Where d is dose ratio

From M, strength of unknown can be calculated

Strength of test =s1 /t 1 antilog of M

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Reliability of this assay is excellent but too time consuming

Lesser in use

3 conc. of each standard & test used in 6 sets (6 doses in each

set) is considered (66=36 doses) in randomized fashion

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DRC of standard drawn on graph paper known as Log doseresponse (LDR) curve

Competitive antagonist addition, lead to shift in LDR curve

to right