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Bioassay Dr. Mayur M. Maybhate JR III, Dept. of Pharmacology IGGMC, Nagpur.

Slide 2:

Introduction Principles of bioassay Applications of bioassay Methodology Classification of bioassay Bioassay of antagonist Human tissue bioassay Bioassay on intact animal Conclusion


Assay Quantitative estimation of drugs or their active constituents Types 1) Physiochemical assay – spectrophotometry, chromatography 2) Immunological assays – radioimmunoassay 3) Microbiological assay 4) Bioassay

Introduction and History of bioassay:

Introduction and History of bioassay Started in late 18 th century by Paul Ehrlich, when he standardized diphtheria antitoxin. Thereafter, it was common practice to standardize any substance through bioassay. Basic step towards drug discovery

Slide 5:

Bio – living material, assay – assessment at laboratory, that means assessment of unknown substance on any living tissue. Comparative assessment of relative potency of a test compound to standard on any living animal or biological tissue .

Characteristics of good assay:

Characteristics of good assay Sensitivity – ability to detect smallest concentration Specificity – ability to detect substance of interest and nothing else Repeatability – Same observations using same instrument and operators over short period of time. Reproducibility – Same observations using different instrument and operator, over longer time periods

Slide 7:

High accuracy, but low precision High precision, but low accuracy Accuracy – Ability of test to hit bull’s eye Precision – Consistency of hitting the target

Slide 8:

Bioassay may be performed on In Vivo In Vitro On intact animal - Isolated tissues - Specific cells - Organisms in culture

Advantages of Isolated tissue experiments over intact animal experiments:

Advantages of Isolated tissue experiments over intact animal experiments As several preparations can be obtained from a single animal Relatively small amount of the test material is required Cheap, less time consuming Interference due to pharmacokinetic factors and Compensatory reflexes is avoided

Choice of tissue for assay:

Choice of tissue for assay • It should give measureable responses with discrimination in the practical range of concentration • It should be able to differentiate between compounds • Tissue should behave similar to human tissue with respect to that receptor and response to the drug • Availability of animals and feasibility • Time consumption - Dosing cycle - Agonist or antagonist is being assayed

Principles of bioassay:

Principles of bioassay A) Comparison of main pharmacological response of unknown preparation to that of standard Its main purpose is determination of amount of substance in relation to functional response of a standard of a same substance Thus, bioassay compares test drugs potency with standard quantitatively

Slide 12:

B) The reference standard and the test sample should have same pharmacological effects and should have same mode of action So that, their log-dose curves run parallel. ( Parallelism must be proved). Hence potency ratios can be compared.

Slide 13:

C) Test solution and reference standard should be compared for their established pharmacological effect using specified pharmacological technique Ach and Histamine can be compared with their test samples for their contractile response on frog rectus muscle or isolated guinea pig ileum resp.

Slide 14:

D) Method selected should be reliable, sensitive, reproducible and should minimize errors due to biological variation and methodology Hence animals should be of same species, sex, weight

Applications of bioassay/ Situation demanding bioassay:

Applications of bioassay/ Situation demanding bioassay 1) When active principle of test drugs is not known but the drug is important and therefore has to be assessed for its biological activity E.g. Antitoxins, vaccines where actual structure is not known but its activity is well established

Slide 16:

2) When test preparation is mixture of chemicals with similar structure but variable biological activity i.e isomers Bioassay will assay only active isomers not the inactive, unlike chemical assay which measures both. E.g. Cardiac glycosides contain Mixture of digitalis, digoxin and gitoxin and ouabanin

Slide 17:

3) Substances with different chemical structure shows similar biological activity 4) Standardization of drug of natural origin whose structure or origin is unpredictable ( Biostandardization )

Slide 18:

5) The chemical assay is not sensitive enough to detect very small quantities of substance If results of chemical and bioassay differ significantly, bioassay results should be taken as final.

Slide 19:

6) Screening of new compound for its biological activity 7) Estimation of biologically active substance like histamine, Ach, 5-HT, adrenaline, PGs, Sub P etc. 8) To estimate EC50, IC50 of a compound



Assembly for recording contraction of isolated tissues:

Assembly for recording contraction of isolated tissues Rudolph Magnus was first to design organ bath Water bath – glass or perspex with electrical thermostat and stirrer for uniform temp Inner organ bath – variable capacity (5-100 ml) for tissue suspension

Slide 22:

Coil – glass or Perspex, connected to lower end of organ bath by rubber tubes, to keep PSS warm before entering into organ bath Writing lever with clamps Rotating drum with Kymograph – for recording activity of tissue

Fixing of tracing:

Fixing of tracing 1) 100 g powdered amber resin or colophony dissolved in 1 ml ethanol ; 10 ml glycerine added to it 2) Granular white shellac dissolved in methanol till it gets saturated and precipitated then allow it to settle for 5-7 days. Take the nonprecipitated solution, store it in cool and dark place.

Example of different levers:

Example of different levers Simple lever (T) Frontal writing lever (P) Heart (starling’s) lever (T) Universal (Brodie’s) lever (T) Gimbal lever (T) Auxotonic lever (P)

Magnification and its adjustment:

Magnification and its adjustment For amplification of response M = Distance from fulcrum to writing point Distance from fulcrum to tied tissue (Fast contracting – low, slow contracting - high)

Type of tissue:

Type of tissue According to tissue obtained According to response by tissue Smooth muscle preparations – Uterus, tracheal SM, ascending and descending colon Slow contracting tissue – frog rectus, stomach fundus, tracheal SM Skeletal muscle – frog rectus Fast contracting tissue – Ileum, uterus, ascending and descending colon Cardiac muscle – frog heart

Types of contraction:

Types of contraction Isotonic – shortening of muscle during contraction that can be recorded under steady load (tension) Isometric - Development of tension during contraction without allowing muscle to shorten Both responses can coincide in muscle. Michelson in 1976 compared quantitatively ratio of EC50 isotonic /EC50 isometric e.g. Frog rectus : 5, GP ileum : 1

Physiological salt solutions (PSS):

Physiological salt solutions (PSS) Often called RINGER solution Different for different tissue preparations > Amphibian tissue – frogs ringer > Mammalian or avian skeletal muscle – Kreb’s ringer > Intestine – Tyrode > Heart – Ringer locke > Rat uterus – De jalon

Slide 30:

Ingredients Tyrode De Jalon Frog Ringer Krebs NaCl 80 90 65 69 KCl 2 4.2 1.4 3.6 Glucose 10 5 20 20 KH2PO4 - - - 1.6 NaH2PO4 0.5 - - - NaHCO3 10 5 2 21 MgSO4.7H2O - - - 2.9 CaCL2 2.64 0.8 1.58 3.70 PSS Composition (salts in g/10 lit)

Role of each ingredient in PSS:

Role of each ingredient in PSS Function Sodium Major ECF cation, maintains electrolyte level in tissue. Make solution isotonic by maintaining osmolarity . Potassium Major intracellular cation, important role in nerve conduction, muscle contraction Calcium Controls excitability of muscle, nerves and glands . (contraction-relaxation coupling ). Maintains integrity and permeability of cell Magnesium Reduce the spontaneous activity of tissues, imp role in neurotransmission in muscle contraction Bicarbonate Acts as buffer Glucose Major nutrient for tissue in invitro set-up


PRECAUTIONS TO BE TAKEN Solution prepared with the help of glass distilled or deionizer water Aeration is important for PSS with oxygen or carbogen (95% O2 + 5% CO2) - Pure O2 may interact with HCO3 in PSS which will cause CO2 loss and PSS becomes alkaline.

Slide 33:

Calcium chloride should be added last , to prevent precipitation or chelation of bicarbonate which makes solution turbid. Error while preparing PSS<1% MgCl2 and CaCl2 are hygroscopic – Use freshly prepared solutions

Slide 34:

Storage of PSS: Can be kept upto 24 hours in refrigerator If storage needed for >24 hrs, avoid glucose and calcium, which should be added at time of experiment pH – Between 7.2-7.4 (if decreases, reduces tonus of tissue) If the solution turns turbid  check pH  usually alkaline  add a few drops of 1N HCl till the solution just becomes clear  Check pH again  proceed if pH is 7.2-7.4

Dissection and treatment of the tissues::

Dissection and treatment of the tissues: Kill the animal by a sharp blow on the head Dissect out the required tissue with minimal handling and immediately place it in the PSS with appropriate aeration In case of intestinal tissue, the contents of the gut can be washed out with the help of pipette containing PSS and held into one of the openings of the selected piece at 45 degree

Care while mounting the tissue:

Care while mounting the tissue Avoid use of metal and red rubber in any portion of the assembly [ except drain] Balance the lever before mounting the tissue Set the temperature of the thermostat as per protocol( if <37 C, tonus of intestine increased, contraction becomes smaller, contraction and relaxation time increased ) Fill the organ bath with PSS

Slide 37:

Start aeration ( not only provides oxygen to tissue, but also stir the bath solution thereby facilitates diffusion of drug added to the bath ) Mount the tissue into the organ bath such that to get desired magnification In case of aorta and tracheal rings preparation, they are cut spirally, later mounted to simulate their natural contraction

Slide 38:

Wash may be done by draining-refilling or overflow » draining-refilling  sagging of tisssue  unsuitable for Isolated tissues with nerves » Overflow  large amount of PSS is required

Care while preparing drug solutions:

Care while preparing drug solutions Must be freshly prepared Stock solutions should be made in DW because some compounds are less soluble in PSS than water but further dilutions should be in PSS Ascorbic acid or HCl should be added to Stock solutions of adrenaline/ NA to give pH 4  stable by minimizing oxidation

Slide 40:

Usually, concentration is described as weight of drug/ml i.e. mg/ml But, it is advised to express concentration in terms of molarity , so its not necessary to specify salt used  more rational and standardized. E.g. Ach available as chloride, bromide, iodide salt  if expressed considering weight, chloride salt seems more active because of its low MW.

Slide 41:

Molarity – No. of moles of a substance in 1 lit solution (1 mole = 1 gram mol. Wt.) To convert mg/100ml to mEq/lit mEq/lit = (mg/100ml ×10 × Valence) / Mol.wt.

Care while performing the assay:

Care while performing the assay • Volume of drug administered each time <10% organ bath volume….temperature fluctuations ?? • Maintain the dose cycle • Check for repeatability • Fill the organ bath upto the same level each time

Slide 43:

Complete adjustment of organ bath, lever, balancing it Select proper tissue, relaxation, attach in organ bath Meantime prepare standard time Start with low dose, make DRC of standard Make DRC with test drugs Select assay method (3,4 point) Calculation

Various isolated tissue preparations:

Various isolated tissue preparations

Slide 46:

Preparation PSS Temp. (degree C) Magnification Used for assay of Guinea pig ileum Tyrode 35-37 5-10 5HT, Histamine, Ach GP tracheal chain Kreb’s 37 12-20 NA, A GP vas deference Tyrode 32-34 4-8 Na, A, Ach, H Rabbit aorta Kreb’s 37 7-10 NA, A Rat ileum Tyrode 37 15 Ach, H Rat colon AC – Kreb’s DC – De jalon 37 25 4 4 NA, Ach, Sub P, AT Rat uterus De jalon 25-36 4 Oxytocin , 5HT, A Rat stomach Tyrode 37 5-16 5HT, Ach

Sensitivity to active substances:

Sensitivity to active substances Preparation 5 HT Ach AT BK NA H Rat As. colon 2× 10 -7 2× 10 -8 2× 10 -9 2× 10 -7 2× 10 -7 10 -5 Rat Stomach 10 -9 2× 10 -8 2× 10 -9 2× 10 -8 2× 10 -7 0 Rat duodenum 2× 10 -8 2× 10 -8 2× 10 -7 2× 10 -9 2× 10 -7 0 GP ileum 2× 10 -8 2× 10 -8 2× 10 -8 2× 10 -7 2× 10 -7 10 -8 Note : Values in gm/ml bath fluid

Dose response curves:

Dose response curves Potency comparison of std. and test always depends on response produced by particular concentration of std. or drug. Obtain DRC with increasing concentration of agonist, starting with lowest dose Instead of plotting graph of dose and % response, its better to go for log dose Vs % response

Advantages of logarithmic transformation:

Advantages of logarithmic transformation Results can be plotted when the doses vary over 1000 fold range, which otherwise is not possible DRC becomes linear in middle part Error is uniformly distributed and independent of dose Has mathematical advantages when two DRCs are compared

Slide 50:

DRC characterized by 3 parameters 1) Maximum or ceiling effect – Gives efficacy , Emax 2) Position of curve – Gives idea about potency of drug. May be expressed as EC50. Lower the value, more potent is the drug OR pD2. 3) Slope of curve – also called regression coefficient Determines error or reliability (precision) of assay

Cumulative Dose response curve:

Cumulative Dose response curve Cumulative DRC of standard obtained by increasing concentrations of drug step by step without washing previous dose Response steadily reaches maximum, doesn’t increase further even after increasing dose Response plotted Vs log dose, and concentration of test can be calculated

Slide 52:

Advantages Disadvantages Simple Can not be adopted for unstable drugs Less time consuming Desensitization can occur Good sensitivity Can’t be done with small organ bath Difference in potency can be calculated Drug antagonism can also demonstrated

Errors in bioassay:

Errors in bioassay Biological variation – errors in response due to some fault in tissue itself Reason Correction Downregulation of receptors Avoid over washing of tissue Loss of sensitivity Change the tissue Lab conditions may vary Should be maintained constant

Slide 55:

Methodological error – error due to faulty method selection or incorrect procedure Reason Correction Lack of standardization of procedure Standardize the procedure beforehand Set-up of apparatus Check every instrument for proper function Tissue isolation and preparation for expt. Minimize the handling and excess cleaning of tissue while mounting Preparation of PSS Follow the procedure, maintain pH Drug preparation and dilution Mix slowly while diluting, avoid vigorous shaking

Types of bioassay:

Types of bioassay Quantal ( Direct end point) Assay Graded assay Bracketing assay Matching assay Interpolation assay Multiple point assay

Quantal response (All or none assay):

Quantal response (All or none assay) In this assay, dose of standard and unknown which provide predetermined ‘all or none’ response, then their potency ratios compared. E.g. Digitalis induced cardiac arrest in cats, insulin induced hypoglycemic convulsions in mice

Slide 58:

The extract containing digitalis (std or unknown) infused into vein of cat till heart stops. Volume of fluid passed into vein noted. (threshold dose) TD50 for test and standard calculated and compared.

Slide 59:

Advantages Disadvantages Drug effects appears rapidly and easily recognized Only toxicity or high dose study possible Effect directly proportional to drug dose Dose ranging study can’t be done Rapid end point detection

Graded response assays:

Graded response assays Responses to various doses of drug are graded and measured Effect of standard and unknown drug measured repeatedly on same tissue. - Matching assay - Bracketing assay - Interpolation method - Multiple point assay

Matching assay:

Matching assay Done when test sample is too small Concentration of unknown which matches in its response with known response of the standard is found by trial and error method From this, potency of two can be found and strength of unknown can be calculated.

Bracketing assay:

Bracketing assay It is also used when test sample is too small. Observed response with a test drug is bracketed between one higher and one lower response of standard. Strength of unknown can be found by simple interpolation of this bracketed response on dose axis

Advantages and disadvantages:

Advantages and disadvantages Advantages Disadvantages Faster Match is subjective Can be completed when amount of test drug available is small Exact match may not always be possible Does not involve complicated calculations No evidence of parallelism/ discrimination [Repeat with a different dose of standard and test]

Interpolation method:

Interpolation method This method depends on assumption of log-dose response curve Log-dose response curve with standard drug obtained. Later 2 or 3 such responses of unknown, which fall between linear portion (25-75%) of LDR curve are obtained by trial and error By interpolation of response at dose axis, and taking the antilog, concentration of unknown can be found.

Multiple point assay:

Multiple point assay The above mentioned method are not ideal because lack of accuracy, sensitivity and involve many methodological errors such as sensitivity error, temp error, dilution error etc. Hence to minimize those errors, multiple point assays preferred. (3, 4 or 6 point assays )

3 point assay:

3 point assay Method depends on Latin square randomization of total 3 responses selected for DRC prepared for standard as well as test drug Can take 25 and 75% or 30-70% of max response to calculate s1 and s2. Select T in between those readings. 3 cycles - (s1,s2,t), (s2,t,s1), (t,s1,s2)

Slide 71:

A] Calculate relative potency of test using the formula M=[ s1/t] X antilog[{(T-S1)/(S2-S1)}X log(s2/ s1)] Where S1 and S2 – mean length of std in mm T – mean length of test in mm s1 and s2 – standard dose which has given the responses S1 and S2 resp. t – test dose which has given response T B] Calculate graphically as for interpolation but express the answer as Relative Potency= antilog [Log t’-Log s1]

Four point assay:

Four point assay Method is same as 3 point assay, only difference is that, 4 responses (2 of standard and 2 of test) from DRC for 16 consecutive Latin square randomization This will further reduce error or variability in response Calculate Strength or relative potency of test using the formula =[ s1 /t1] X antilog[{(T2-S2+T1-S1)/ (S2-S1+T2-T1)} X log(s2/ s1)]

Slide 74:

Calculating the error = Actual concentration-Observed Concentration X 100 Actual concentration Acceptable error in agonist assay <10%

Bioassay of antagonist:

Bioassay of antagonist Several types of antagonism 1. Physical antagonism 2. Chemical antagonism 3. Physiological antagonism 4. Receptor level antagonism – Competitive or noncompetitive

Slide 76:

In practical pharmacology, identification of antagonist is limited to competitive or noncompetitive or physiological antagonist only. Antagonist – any drug which can completely or partially block the response of an agonist i.e. Max affinity, but no intrinsic activity

Slide 77:

2 points should be kept in mind a) To check whether the antagonism is surmountable by increasing the concentration of agonist or not b) To check whether the antagonism is reversible or not - If yes, the antagonism is likely competitive.

Slide 78:

Experimentally, antagonism is divided into 2 groups ; • Preventive: Antagonist  Agonist • Curative: Agonist  Antagonist

Determination of Dose ratio:

Determination of Dose ratio Effect of antagonism can be measured in terms of dose ratio Dose ratio = EC50 after antagonist/ EC50 before antagonist Gives factor by which agonist conc. To be multiplied to get given response in presence of antagonist Higher the dose ratio, more specific is the antagonist

Calculating dose ratio [Competitive antagonist]:

Calculating dose ratio [ Competitive antagonist] • DRC of agonist • Add the relevant dose of Antagonist  allow to achieve equilibrium • DRC of agonist in the presence of antagonist • Plot the 2 DRCs as %max Vs Log Dose of agonist • Calculate graphically the Equi-effective dose of agonist in the presence or absence of antagonist

Slide 81:

Dose ratio = antilog[Log a2-Log a1]

Determination of IC50 value:

Determination of IC50 value Simplest way of expressing antagonist quantitatively Concentration of antagonist produces 50% inhibition of effect of agonist IC 50 gives a measure of potency of the antagonist

Calculating IC50:

Calculating IC50 • DRC of agonist • Add a dose of antagonist to the organ bath  allow to achieve equilibrium • Add a dose of agonist from the 25%-75% portion of the DRC in the presence of antagonist • Note the response • Wash out the antagonist • Check for regaining of sensitivity of the tissue to agonist • Add another dose of antagonist [> or < than the previous dose depending upon the extent of inhibition produced

Slide 84:

• Repeat the same dose of agonist in the presence of antagonist • Plot the 2 points on the graph paper as %inhibition Vs Log Dose of antagonist

Methods to identify competitive antagonist:

Methods to identify competitive antagonist Parallel shift of DRC Double reciprocal plot Lineweaver and Burk Schild plot method and pA2, pA10 values

Shift of DRC:

Shift of DRC

Slide 87:

Lineweaver Burk plot [Double reciprocal plot] - Here, inverse of response is plotted against inverse of dose of agonist - Responses are plotted in absence or presence of antagonist competitive inhibition – V max remains same, so its reciprocal So, Y intercept of drug response of both is same, with different slopes and X intercepts

Schild plot method and pA2 and pA10 values:

Schild plot method and pA2 and pA10 values Potencies of competitive antagonist are expressed as pA2 values Defined as negative logarithm to base 10 of antagonist concentration in molar units corresponding to dose ratio of 2 i.e. concentration that produces 2 fold shift in agonist DRC K d (dissociation constant of antagonist for receptor) is the other form of expressing potency of competitive antagonist

Slide 89:

The pA 2 is calculated by extrapolating the value on the x-axis when y=0

Slide 90:

The slope of the Schild plot gives information about the nature of the antagonist i.e. whether or not it is competitive binding and information on the cooperativity. The steepness of slope depends upon both the equilibration time and the degree of antagonism For Competitive antagonist – slope is 1. And (pA2-pA10) value is 0.95 (0.8-1.2)

Identification and assay of unknown substance:

Identification and assay of unknown substance Screen the unknown by multiple isolated preparations depending on availability Later, presence of substance can be confirmed by use of specific antagonists .

In vitro screening of active substances present in biological fluids and tissues:

In vitro screening of active substances present in biological fluids and tissues Tested on GP ileum Tested on Rat duodenum Tested on Rat ascending colon Contraction 5-HT, Ach, Angiotensin, Bradykinin, Histamine Contraction 5-HT, Ach, Angiotensin Contraction 5-HT, Ach, Angiotensin Histamine Relaxation or no effect NA, Adrenaline Relaxation Bradykinin No effect Histamine The identification of the compound is finally confirmed by use of respective antagonist

Interpretation of results obtained by GP ileum:

Interpretation of results obtained by GP ileum Inference Atropine (0.1 mcg/ml) Muscarinic Hexamethonium (100 mcg/ml) Ganglionic stimulant Mepyramine (0.01 mcg/ml) Histaminic Methylsegide (0.5 ng /ml) Serotonin like None of above Direct SM stimulant peptide If the compound stimulates ileum which is antagonized by

Slide 94:

If a compound antagonizes the ileum stimulated by Acetylcholine Atropine like Histamine Antihistaminic Serotonin Antiserotonergic Nicotine or DMPP Ganglionic blocker All the above Papaverine like or sympathomimetic

Human tissue bioassay:

Human tissue bioassay Limitation of animal tissues: – Species variable  cannot predict actual outcome in relation to the human Use of human tissue and use of cell lines with close precision to human Tissue difficult to obtain because of Ethical limitations, and difficulty in standardization small experimental window

Slide 96:

Human tissues that can be used in some industrial drug innovation protocols • Veins [obtained from surgery on varicose veins] • Tissues like larger blood vessels obtained during amputation • Organs removed during transplantation/ tumor surgeries/procedures requiring resection • Tissues collected during post mortem

Bioassay on intact animals:

Bioassay on intact animals Generally, Dogs, cats are used. Recording of blood pressure in anesthetized animal is one of the standard method for assay of drugs. Suitable Anasthetiac  Carotid A. cannulated Trachea cannulated  Femoral vein cannulated Drugs given

Drugs assayed by dog BP method:

Drugs assayed by dog BP method Adrenaline – all adrenergic receptors Noradrenaline – No β 2 action Acetylcholine Histamine

Identification of unknown by testing on dog BP:

Identification of unknown by testing on dog BP Series of standard tests carried out on BP of anesthetized animal Normal response brackets 1) Electrical stimulation of vagus 2) Inj. Ach (0.5-5 mcg/kg) 3) Inj. NA (1-5 mcg/kg) 4) Inj. Adrenaline (1-5 mcg/kg) 5) Occluding both carotid arteries for 30 sec.


Conclusion Bioassay is for determining quantitative relationship between dose and degree of response it produces Vast applications in pharmacology


References MN Ghosh , Fundamentals of Experimental Pharmacology; 4;106-170;2008 Bikash Medhi , Practical manual of experimental and clinical pharmacology;1;45-70;2010 HL Sharma, KK Sharma , Bioassay, Principles of Pharmacology;2;927-29;2011

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