bioassay & biostandardisation
DESCRIPTION
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BIOASSAY & BIOSTANDARDISATION
Dr. Anoosha Bhandarkar
Post Graduate
Department of Pharmacology
SDM Medical College 21-07-2011
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OVERVIEW Experimental Pharmacology
Assay Bio assay
When bioassay? Applications Principles Types of Bio assay
Requirements Merits & demerits
Different tissues used Human tissue bioassay
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EXPERIMENTAL PHARMACOLOGY
Aims:
To find out therapeutic agents suitable for human
use
To study MOA & site of action
To study toxicity of drugs – acute , subacute, chronic
Types : qualitative ( analyse activity) & quantitative
( assay activity)
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BIOSTANDARDISATION
Introduced in 1920s by Paul Ehrlich : bio-
standardization of Diphtheria antitoxin by his “side-
chain” theory of immunity.
Defn: Comparison and adjustment of the strength of
the sample with that of the standard under
controlled conditions
Necessary to regulate the doses of crude extract.
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ASSAY Definition:
Quantitative estimation of drugs or of their active
constituents
Types
Physico-Chemical Assays: eg. Salicylates, Sulfonamides
etc..
M/commonly used procedure . Eg. Spectrophotometry,
Chromatographic & mass spectrometric techniques
Immuno - Assays : RIA , immunoradiometric assays –
for protein hormones & drugs
Biological Assays (Bioassay)
Microbiological assay
Radio-receptor assays: principles of Bioassay +
Radioimmuno assay
SELECTION OF METHOD:-
PHYSICAL
• PHYSICAL PROPERTY LIKE COLOUR , FLOURESCENCE
PHYSIO-CHEMICAL
• PHYSICAL PROPERTY & CHEMICAL REACTION
BIOLOGICAL
• BIOLOGICAL PROPERTY USED TO ESTIMATE ACTIVITY
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BIOASSAY
Estimation of concentration or potency of a
substance/drug from the magnitude of its main
biological response.
Main pharmacological effect of the drug under test is
compared with that of standard drug & their potency
ratios are compared quantitatively
Classified : Qualitative & Quantitative
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.
Qualitative bioassays : used for assessing the physical effects of a substance that may not be quantified,
- abnormal development or deformity. Eg. Arnold Adolph Berthold’s experiment on castrated chickens
Quantitative : estimation of the concentration or
potency of a substance by measurement of the biological response that it produces.
Analyzed using the methods of biostatistics.
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Whole animal Isolated tissue Cells for in vitro study
Micro-organisms Observation of pharmacological effects on : • Isolated tissues
or cells for invitro study
• Micro-organisms• Whole animals –
singly/groups
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TISSUE 1. Whole animal
SUBSTANCE a. Vasopressin
b. Estrogens
c. Vit D
d. Insulin
e. d-tubocurarine
RESPONSE ELICITED - Anaesthetised rat for rise in B.P - Hydrated rat for reduction in U/O
- ovariectemised female rat for vaginal cornification
- Rat, alleviation of the rachitic state
-- mice, hypoglycemic convulsions or death
-- Rabbit, head drop d/t paralytic relaxation of skeletal muscles
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2. Isolated tissue
a. Ach
b, Histamine
c. Adr
d. Oxytocin
e. 5HT
-Frog, rectus muscle contraction
- Guinea pig ileum, contraction
-Rat uterus in diestrus, relaxation
- Rat uterus estrogen primed, contraction
- Gastric fundus contraction
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TISSUE
cells dispersed in a suitable medium
RESPONSE ELICITED
IN VITRO measurement of
concentration of Plasma LH
by Levels of testosterone
synthesis by isolated Leydig
cells of testes
Micro organisms Vitamin B12: Growth of Euglena
gracilis
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INDICATIONS FOR BIOASSAY Chemical composition is not known but has a specific
biological action. Eg. LATS
Chemical assay method is too complex/ insensitive. Eg. Adr, His can be bioassayed in micrograms
Drugs differ in composition having same pharmacological action. Eg. Digitalis glycosides
When active principle is unknown/ cannot be easily isolated.
Eg. Peptide hormones- insulin, GH
BIOASSAY GENERALLY EMPLOYED IN.. Estimate concentration/potency of known active principle
in tissue extract.e.g..insulin; body fluids – Serotonin
Estimate pharmacological activity of new/ chemically
undefined substance – agonists / antagonists on a tissue
Comparing competitive v/s non-competitive antagonist
(antagonists assay)
Measure drug ED50, LD50 & adverse effects
Investigate function of endogenous mediators- Tyramine
Subtype of receptors & Tissue selectivity of drug
Diagnostics, toxicity studies , research.
IT IS ONLY METHOD UNDER FOLLOWING CONDITIONS..
If active principle of drug is unknown. e.g. insulin.
Chemical method not available.
Chemical composition not known.
Quantity of the sample is too small. Eg. micrograms
Purification for chemical assay not possible.
As quantitative part of screening procedure.
Investigate function of endogenous mediators- PGs, EDRF,
hypothalamic factors
Measure drug toxicity & adverse effects
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STANDARD CHEMICALS
Representative of a substance serves as basis for comparative
measurement of activity.
Internationally agreed upon standards are necessary – compare
potency
A stable standard solution has to be employed for comparison
Standards for sera held at State Serum Institute, Copenhagen
- National Inst.for Med Research(UK)
In India Standard chemicals maintained & distributed by:
Central Drug Research Laboratory, Kolkata.
Central Research Institute, Kasauli
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PRINCIPLES OF BIOASSAY
•
Comparison of the main pharmacological response of the
unknown preparation with that of the standard
When pure substance unavailable – standardised prep. of
hormones/ natural products used
Ref standard & Test sample should as far as possible identical,
viz. pharmacological effects, mode of action, LDR run parallel &
potency ratios conveniently compared
Specific effect produced by the active principle must be the same
in all animal species
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Certain quantity of drug produces same degree of response in
same animal or animals of same species under identical
conditions. Eg. Adr same degree of rise in BP
Compared for their established pharmacological effect using a
specified pharmacological technique . Eg. ACh on frog rectus &
His on guinea pig ileum/tracheal ring chain prep.
Assayed activity should be the activity of interest
Method should minimise /estimate as far as possible the error due
to biological variation.
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REQUIREMENTS ..
High sensitivity
Specificity. Eg. Guinea pig ileum atropinised for the
bioassay of histamine
Reproducibility- response with the dose shoild
remain the same
Accuracy
Stability – tissue has to stay “bioassay-fit”
Easy availability of animals
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TYPES OF BIOASSAYS Indirect assays : potency of sample estimated by
comparing LDR curve of sample with the similar curve of the standard
Eg. Bioassay of crude ergot preps. Ergot preparations injected in the whiteleghorn
cock vasoconstriction bluish discoloration of comb
Direct assays : dose of the sample required to elicit a particular pharmacological effect (ED50 or LD50) is measured . Eg. Death in guinea pigs
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DIRECT ASSAYS
1. Quantal assay: direct end-point
assay
The dose of the std &
that of the unknown producing a
Predetermined “all or none” response is
measured & potency ratios compared.
Dose is known as “tolerance”/”threshold”
dose
Ratio of TD50 sample/ standard relative
potency of sample
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TD50 (threshold dose producing cardiac arrest in 50% of the subjects) is then calculated for standard (log x) & unknown (log x1)
Toxicity(LD50) & potency(ED50) ratios are calculated & compared
Strength of the unknown calculated
Eg.
D-tubocurarine induced head drop in rabbits
Insulin induced hypoglycemic convulsions in mice
Calculation of LD50 of drugs in mice or rats
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2. Graded Response Assays [mostly on tissues]
Graded responses to varying doses of a drug
Proportionate increase in responses with increase in dose
Effect of both , the standard drug & the Unknown measured
repeatedly on the same tissue
Eg. Bioassay of histamine on guinea pig ileum
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BASIC REQUIREMENTS FOR BIOASSAY
Instrumentation
Physiological salt solution(PSS)
Procedures & drugs – to render the animals
unconscious
Tissue – isolated / whole
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APPARATUS Outer water bath of perspex/glass Inner organ bath {single or multiple}- glass. 15-
100ml Tissue holder cum O2 tube Glass coil connected to organ bath, mariott’s bottle Thermostat Electrical stirrer Writing lever
Platinum electrodes embedded in plastic for innervation of nerves
Recording drum, Adjustable clamps, holders, grips, X blocks, dissection instruments etc.
Frontal lever, Gimbal lever Sprung lever
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SALT SOLUTION
Frog ringer: Frog heart & tissue, Ringer’s solution: Mammalian isolated heart & other tissuesTyrode’s: Mammalian smooth muscle, Kreb’s: Mammalian isolated organ specially for nerve responses, DeJalon : No Mg2+/PO4+ ions
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PROCEDURES TO RENDER ANIMALS UNCONSCIOUS
Mice, rats, guinea pigs & rabbits : stunning ( crushing the skull ) followed by cutting the throat for bleeding
Frogs : - Chilling to 4°C until state of immobility reached - Pithing – single & double (unless & otherwise) Lab. anaesthetics used:
Barbiturates mostly used.
Eg. Pentobarbitone sodium(NEMBUTAL), phenobarbitone sodium
Produces anesthesia in dose of 35-45mg/kg. i.p./i.v.Duration 45-60
min.
Others : chloralose (80-100mg/kg,ip or iv)& urethane (1-1.5g/kg,ip or
iv), paraldehyde (2-2.5mg/kg i.m / i.v), 20% MgSO4 (5ml/kg i.v )
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Tissue Use-Assay Cycle Remarks
Frog Rectus abdominusFrog Ringer
1. NM blockers2. Nm receptor agonists3. Analyzing AntiChE
activity/Susceptibility to ChE
Drug 90sRest- 4-4.5 min
30 min relaxation prior to startSturdy, slow contracting
Dorsal muscle of LeechFrog Ringer or Ringer Locke soln-5:7 dil
1. NM blockers2. Nm receptor agonists3. Analyzing AntiChE
actvity/Susceptibility to ChE
Drug 90sRest- 13.5 min
Delicate tissueMost sensitive to Ach after addition physostimine
Guinea pig ileumTyrode soln.
Agonist and Antagonist onMuscarinic, Histaminic, 5HT, nicotinic, Bradykinin receptor
Drug 30sRest- 2.5 min
Delicate tissue, Non-specific, May show spontaneous activity
Rat Phrenic nerve diaphragmKreb’s solution
NM blockersLocal anaesthetics- Nerveblock
Drug -3 min,Rest- 6 min
Care of the nerveDrain by overflow
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Tissue Use-Assay Cycle Remarks
Rabbit JejunumTyrode
α adrenergic agonist & antagonist
Drug 30sRest- 150 s
Spontaneous pendular movements inhibited by α adrenergic agonist
Finkelman Preparation: Rabbit jejunum withmesentery
α adrenergic antagonistsAdrenergic neurone blocking agentsNerve block local anaesthetics
Slow rate2-4/secParasymp.Fast rate30-50/secSymp. stimulation
Mesentery contains sympathetic &Parasympathetic nerves
Rat FundusKreb’s solution
5HT agonist & antagonists Drug 90sRest- 4.5 min
Relax for 30 min before experiment
Rat UterusDe Jalonslow Ca++
Temp: 30
Oxytocin & its antagonist, Ach, β adrenergic agonist and antagonist
Drug 30 sRest- 2.5 min
Pre-treatment with stilbesterol
Guinea pig-Tracheal chainKreb’s solution
Histamine, ACh, 5HT, β adrn agonist & antagonist
Drug 5 minRest- 10 min
Special manner of tying the ring
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METHODS OF DOING GRADED BIO ASSAY Matching bioassay
Bracketing assay
Interpolation method
Multiple point bioassay
Three point bioassay
Four point bioassay
Six point bioassay
Not reliable
Not consider sensitivity change w.r.t
time, timing of doses, variations in
methods of application of drugs &
Few dose analysis
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MATCHING BIOASSAY
Employed for small sample size
1. Firstly responses of the test of a particular dose is
taken
2. It is matched with the dose of the standard (whose
strength is known) by trial & error method
3. Done till a closed matching is observed.
4. Corresponding concentration calculated.
5. Potency ratio of the two can be approximately found
& strength of the unknown test solution can be
calculated
6. Eg. histamine bioassay, posterior pituitary assay on
the rat uterus
go
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BRACKETING METHOD
Used when test sample is too small
1. Response of test is bracketed b/w two responses
(greater & smaller) of standard substance.
2. Strength of unknown found by simple
interpolation of this bracketed response on the
dose axis
Precision & reliability is poor.
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MATCHING/ BRACKETING
Advantage: Faster Can be completed when amount of test drug
available is small Does not involve complicated calculations
Disadvantage Match is subjective Exact match may not always be possible No evidence of parallelism/ discrimination Does not permit calculation of variation.
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INTERPOLATION METHOD
1. Log dose response curve of std. is established
with atleast 4 submaximal concentrations
2. 2-3 responses of test is recorded.
3. Select dose responses that lie on LINEAR
PORTION of LDR curve of standard
Strength of unknown found by interpolation of these on dose axis &
taking antilog Advantage:
Faster
Can be completed when amount of test drug available is small Disadvantage:
No evidence of parallelism/discrimination
Does not permit calculation of variation/ precision
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THREE POINT BIOASSAY [2+1 DOSE ASSAY]
Fast & convenient procedure
LDR curve plotted with varying conc. of std & test solution
2 standard, s1& s2 [1:2 dose ratio] from linear part of LDR & 1 test response [between s1 & s2]are taken
Record 4 sets data
• Latin square: Randomisation reduces error
Plot mean responses of S1, S2 and T against dose.
Calculate Log Potency ratio
• M = [ (T –S1) / (S2-S1) ] X log d [d = dose ratio]
Strength of unknown calculated as
• Strength = s1/t (antilog of M)
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CHART CALCULATION
Response
Hts of contractn 1 (mm)
2 3 4 Mean (mm)
Dose(ml)
Dose ratio
S1 35 40 40 45 40 0.1 s2/s1 = 2
S2 75 80 80 85 80 0.2
T 55 60 60 65 60 0.25
Log potency ratio M = [ (T –S1) / (S2-S1) ] X log d where [d = dose ratio=2]M= 0.150; Strength of the unknown = s1/t * (antilog of M) = 0.5652 × potency of std.
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FOUR POINT BIOASSAY
Most common method used; 2 standards (s1,s2) & 2 test (t1,t2) responses
• M= {(T1-S1+T2-S2) / (S2-S1+T2-T1)} * log d. d = dose ratio
Two responses of standard & test should lie on linear portion of CRC in ratio of 1:2
4 sets of bioassay are done using Latin square design
Plot mean responses of s1, s2 & t1, t2 against dose
Horizontal separation: log potency ratio(M) of the conc. of T & S
Strength of T = (s1/t1) * antilog of M
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CHART CALCULATION
RESPONSE
Hts of contraction 1 (mm)
2 3 4 Mean ( mm )
Dose
Dose ratio
S1 35 40 40 45 40 0.1 S2/S1
S2 75 80 80 85 80 0.2 t2/t1=2
T1 40 45 45 50 45 0.15
T2 80 85 85 90 85 0.3Log potency ratio M= {(T1-S1+T2-S2) / (S2-S1+T2-T1)} * log d d = dose ratio = 2
M = 0.037; Strength of unknown soln T = (s1/t1) * antilog of M = 0.726 × potency of std.
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SIX POINT BIOASSAY (3 + 3 ASSAY)
Reliability is excellent Time consuming Three concentration of standard & test are used. 6 sets of experiments using 6 doses in each set
6*6 = 36 doses in latin square design
Recent applications:- Microbiological assay of vit B12- Analgesic assay of sublingual buprenorphine & i.m
morphine- Diazyme’s cystatin-C assay –emerging marker–renal
disease- Comparitive assays of Erythropoietin standards
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CUMULATIVE DOSE RESPONSE CURVE
• C-DRC is obtained when the drug is added with increasing concentration without washing the previous dose• Tissue sensitivity, tachyphylaxis, antagonist assays
Resp
onse
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MERITS DEMERITS
Biological products like toxin,anti toxin,sera can be conveniently assayed.
Key problem is variability in response.Always not reproducible
Measure minute (nano & Pico mole) quantities of active substances.
Large number of animal to be used. Expertise in experimental design, execution of assay & analysis of data required.
Can detect active substance without prior extraction or other treatment.
Tachyphylactic responses of substance being assayed.
More chemicals in single animal tested
Expensive & time consuming.
Unwanted effects on other system avoided
Time related changes in sensitivity of test organ.
No neuronal response / reflex mechanism
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Antagonists assay Competitive antagonism
• Isolated rabbit aortic strip• Cumulative DRC for NA before &
after Phenoxybenzamine• Max response can be obtained
with increase dose• Parallel shift of curve to right side
with antagonism
Non-Competitive antagonism• Isolated guinea pig ileum• Cumulative DRC of Histamine
before & after Phenoxybenzamine
• Max response is not achieved even with higher dose of Ag
• Flattening of the curve
Physiological antagonism- Cholinergic v/s adrenergic
• Isolated rat colon• Contractile response to
carbachol inhibited by Adr
Non-Specific antagonism• Isolated rat colon• Contractile response to
carbachol inhibited by Papaverine
DRC- Dose response curveNA – Nor-AdrenalineADR- AdrenalinePhenoxybenzamine- Non selective α antagonistCarbachol – cholinergic agentPapaverine- direct smooth muscle relaxant property
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HUMAN TISSUE BIOASSAY Limitation of animal tissues:
Species variable: cannot predict actual outcome in relation
to human
Use of human tissue
use of cell lines with close precision to human
Human tissues that can be used
Veins [obtained from surgery on varicose veins]
Larger blood vessels obtained during amputation
Organs removed during transplantation/ tumor surgeries/
procedures requiring resection/ aborted foetus
Tissues collected Post mortem
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Interestingly, clinical trials for assessing drug effects in humans involve similar principles as bioassays in animals!!
Environmental bioassays for effluent toxicity tests of sewage & industrial wastes is a must for municipal sewage Rx plants at U.S
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REFERENCES
Sharma & Sharma. Principles of Pharmacology. Revised
ed. 2011
MN Ghosh. Fundamentals of experimental
Pharmacology.4th ed. 2008
Pharmacology & pharmacotherapeutics by Satoskar &
Bhandarkar, revised 21st edition
Internet search