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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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INSTRUMENTATION

Rudolph Magnus-1904

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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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BRACKETING METHODback

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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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THREE POINT BIOASSAY [2+1 DOSE ASSAY]

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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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FOUR POINT BIOASSAY

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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