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7/31/2019 Bio as Say 1
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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