BIOASSAY seminar

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Bioassay & Biostandardisation:

Bioassay & Biostandardisation Dr. Anoosha Bhandarkar Post Graduate Department of Pharmacology SDM Medical College 21-07-2011 1

Overview:

Overview Experimental Pharmacology Assay Bio assay When bioassay? Applications Principles Types of Bio assay Requirements Merits & demerits Different tissues used Human tissue bioassay 2

Experimental pharmacology:

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) 3

bIOSTANDARDISATION:

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. 4

Assay:

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 5

Selection of Method:- :

Selection of Method:-

Bioassay:

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 7

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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. 8

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10 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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11 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 12

Indications for Bioassay:

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 13

Bioassay generally employed in..:

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 ED 50, LD 50 & 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..:

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

Standard chemicals:

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) 16 In India Standard chemicals maintained & distributed by: Central Drug Research Laboratory, Kolkata. Central Research Institute, Kasauli

PRINCIPLES OF BIOASSAY:

PRINCIPLES OF BIOASSAY 17 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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18 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.

REQUIREMENTS ..:

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 19

Types of bioassays:

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 20

direct assays:

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 21

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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 23

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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 24

Basic requirements for bioassay:

Basic requirements for bioassay Instrumentation Physiological salt solution(PSS) Procedures & drugs – to render the animals unconscious Tissue – isolated / whole 25

Instrumentation:

Instrumentation 26 Rudolph Magnus-1904

Apparatus:

Apparatus Outer water bath of perspex /glass Inner organ bath {single or multiple}- glass. 15-100ml Tissue holder cum O 2 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. 27 Frontal lever, Gimbal lever Sprung lever

Salt solution:

Salt solution 28 Frog ringer : Frog heart & tissue, Ringer’s solution: Mammalian isolated heart & other tissues Tyrode’s : Mammalian smooth muscle , Kreb’s : Mammalian isolated organ specially for nerve responses, DeJalon : No Mg2+/PO4+ ions

Procedures to render animals unconscious:

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 ) 29

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Tissue Use-Assay Cycle Remarks Frog Rectus abdominus Frog Ringer NM blockers N m receptor agonists Analyzing AntiChE activity/Susceptibility to ChE Drug 90s Rest- 4-4.5 min 30 min relaxation prior to start Sturdy, slow contracting Dorsal muscle of Leech Frog Ringer or Ringer Locke soln-5:7 dil NM blockers N m receptor agonists Analyzing AntiChE actvity /Susceptibility to ChE Drug 90s Rest- 13.5 min Delicate tissue Most sensitive to Ach after addition physostimine Guinea pig ileum Tyrode soln. Agonist and Antagonist on Muscarinic , Histaminic, 5HT, nicotinic, Bradykinin receptor Drug 30s Rest- 2.5 min Delicate tissue, Non-specific, May show spontaneous activity Rat Phrenic nerve diaphragm Kreb’s solution NM blockers Local anaesthetics- Nerve block Drug -3 min, Rest- 6 min Care of the nerve Drain by overflow 30

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31 Tissue Use-Assay Cycle Remarks Rabbit Jejunum Tyrode α adrenergic agonist & antagonist Drug 30s Rest- 150 s Spontaneous pendular movements inhibited by α adrenergic agonist Finkelman Preparation: Rabbit jejunum with mesentery α adrenergic antagonists Adrenergic neurone blocking agents Nerve block local anaesthetics Slow rate 2-4/sec Parasymp . Fast rate 30-50/sec Symp . stimulation Mesentery contains sympathetic & Parasympathetic nerves Rat Fundus Kreb’s solution 5HT agonist & antagonists Drug 90s Rest- 4.5 min Relax for 30 min before experiment Rat Uterus De Jalons low Ca ++ Temp: 30 Oxytocin & its antagonist, Ach, β adrenergic agonist and antagonist Drug 30 s Rest- 2.5 min Pre-treatment with stilbesterol Guinea pig-Tracheal chain Kreb’s solution Histamine, ACh, 5HT, β adrn agonist & antagonist Drug 5 min Rest- 10 min Special manner of tying the ring

Methods of doing Graded bio assay:

Methods of doing Graded bio assay Matching bioassay Bracketing assay Interpolation method Multiple point bioassay Three point bioassay Four point bioassay Six point bioassay 32 Not reliable Not consider sensitivity change w.r.t time, timing of doses, variations in methods of application of drugs & Few dose analysis

Matching bioassay:

Matching bioassay Employed for small sample size Firstly responses of the test of a particular dose is taken It is matched with the dose of the standard (whose strength is known) by trial & error method Done till a closed matching is observed. Corresponding concentration calculated. Potency ratio of the two can be approximately found & strength of the unknown test solution can be calculated Eg . histamine bioassay, posterior pituitary assay on the rat uterus 33 go

Bracketing method:

Bracketing method Used when test sample is too small Response of test is bracketed b/w two responses (greater & smaller) of standard substance. Strength of unknown found by simple interpolation of this bracketed response on the dose axis Precision & reliability is poor . 34

Bracketing method:

Bracketing method 35 back

Matching/ Bracketing:

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. 36

Interpolation method:

Interpolation method Log dose response curve of std. is established with atleast 4 submaximal concentrations 2-3 responses of test is recorded. 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 37

Three point bioassay [2+1 dose assay]:

Three point bioassay [2+1 dose assay] 38

Three point bioassay [2+1 dose assay]:

Three point bioassay [2+1 dose assay] 39

Chart calculation:

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 40 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.

Four point bioassay:

Four point bioassay 41

Four point bioassay:

Four point bioassay 42

Chart calculation:

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 0.3 43 Log 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.

Six point bioassay (3 + 3 assay):

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 44

Cumulative Dose Response Curve:

Cumulative Dose Response Curve 45 C-DRC is obtained when the drug is added with increasing concentration without washing the previous dose Tissue sensitivity, tachyphylaxis , antagonist assays Response

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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 46

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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 curve NA – Nor-Adrenaline ADR- Adrenaline Phenoxybenzamine - Non selective α antagonist Carbachol – cholinergic agent Papaverine - direct smooth muscle relaxant property 47

Human tissue bioassay:

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 48

Slide 49:

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 49

References:

References Sharma & Sharma. Principles of Pharmacology. Revised ed. 2011 MN Ghosh. Fundamentals of experimental Pharmacology.4 th ed. 2008 Pharmacology & pharmacotherapeutics by Satoskar & Bhandarkar , revised 21 st edition Internet search 50