Bioassay

learning objectives: What is assay, definition of bioassay, its advantages & disadvantages ,principles ,types,examples and current status of bioassay.

1. Bioassays Dr U ma Advani Assistant professor pharmacology SMS Medical College, Jaipur

2. Learning objectives What is assay ?Types of assays What is bioassay Indications of bioassay Advantages & Disadvantages of bioassay Principles of bioassay Types of bioassay Examples of bioassay Current status of bioassay Summary

3. What is assay? An assay is a procedure in molecular biology for testing and/or measuring the activity of a drug or biochemical in an organism or organic sample

4. Types of Assays Chemical Assays: Spectrophotometry Spectrofluorimetry Chromatography Immunoassays Biological assays

5. What is bioassay : Bioassay Estimation of the conc. / potency of a substance by measuring its biological response in living systems i.e. Observation of pharmacological effects on Living tissues or cells Microorganisms Intact animals

6. Indications for Bioassay Active principle of drug is unknown Active principle cannot be isolated, e.g. insulin, posterior pituitary extract etc. Chemical method is either Not available If available, too complex, Insensitive to low doses e.g. Histamine can be bioassayed in microgram conc.

7. …Contd. Unknown Chemical composition, e.g. long acting thyroid stimulator. Chemical composition of drug variable but has same pharmacological action e.g. Cardiac glycosides isolated from different sources, Catecholamines etc.

8. Applications of bioassay Standardization of drug Estimation of biologically active substances like acetylcholine, Adr , NAdr in body fluid Screening of new compounds for biological activity Diagnosis and research Estimation of LD50 and ED50

9. Advantages of bioassay Can detect very small concentration of the substance More reliable than chemical method as it is done on a living organism/ tissue.

10. Disadvantages of bioassay Less accurate More laborious Expensive

11. Principles of Bioassay All the bioassay must be comparative against a standard drug or preparation The standard and the new drug should be as far as possible identical to each other The degree of pharmacological response produced should be reproducible under identical conditions i.e. Adr . shows same rise in BP in the same species under identical conditions: wt, age, sex, strain / breed etc

12. …Contd. The reference standard must owe its activity to the principle for which the sample is being bioassayed Activity assayed should be the activity of interest The method should be able to estimate the error due to biological variations Bioassay might measure a different aspect of the same substance compared to chemical assay i.e. Testosterone & metabolites

13. Types of bioassay

14. Methods of bioassay in agonist

15. Methods for antagonists Commonly used method is simple graphical method Two responses of the same dose of agonist (sub maximal giving approximately 80% of the maximum response) are taken The minimum dose of standard antagonist is added in the bath and then the response of the same dose of agonist is taken in presence of antagonist The higher dose of standard antagonist is added and responses are taken as before The percentage inhibition is calculated, plotted against log dose of antagonist and the concentration of unknown is determined

16. Methods for bioassays

17. Methods for bioassay Quantal Assays [ Direct endpoint ] Elicits an ‘All or None’ response in different animals The threshold dose producing a positive effect is measured on each animal and the comparison between the average result of two group of animals is done Eg bioassay of digitalis on cat Drug is infused till the heart stops beating & BP falls to zero The volume of infused is recorded Two series are done one with standard dose & other with test dose Conc. of the unknown= (threshold dose of the standard/ threshold dose of the test) X conc. of standard

18. Contd… e.g . Calculation of LD50 in mice or rats Digitalis induced cardiac arrest in guinea pigs and cat Hypoglycemic convulsions in mice. Digitalis induced head drop in rabbits

19. Contd… Percentage of positive effect measured A quantal response is obtained & the percentage of the positive effects at each dose is computed. Now the unknown is compared to the standard with respect to potency in causing the quantal effects Eg LD50 estimation in toxicity testing The drug/ poison in different doses is injected into animals and percentage of mortality is determined Next the percentage of mortality is plotted against the dose and a curve is obtained and the dose causing 50% mortality is thus obtained Other examples include assays of insulin

20. …Contd. Graded Response Assays [mostly on tissues] Graded responses to varying doses Unknown dose response measured on same tissue

21. Methods of Bioassay Cross over test The effect of the drug is measured more than once in the same animal i.e on day 1, 1 st set receives standard dose while the second set receives unknown Now on another day, 1 st set receives the unknown while the first set receives the standard The average effect for each preparation for both the stages is calculated & the results are interpreted by drawing a log DRC Eg . Assays of insulin on rabbit It increases the accuracy

22. …Contd. Matching / Bracketing: Const dose bracketed with varying doses of standard till exact match is obtained The conc. of unknown= (dose of standard/ dose of the test) X conc. of the standard Used when test sample is too small i.e. Histamine on guinea pig ileum, Ach on frog rectus , Posterior pituitary on rat uterus

23. Matching method

24. Contd… Disadvantages Inaccurate & margin of error difficult to estimate Match is purely subjective

25. …Contd. Multiple Point Assays 3 point assay 4 point assay 6 point assay 8 point assay

26. 3 point assay [2+1 dose assay] Fast & convenient Procedure [e.g. Ach bioassay] Log dose response [LDR] curve plotted with varying conc of std Ach. Select two std doses s1& s2 [ in 1:2 dose ratio] from linear part of LDR Choose a test dose T between S1 & S2

27. …Contd. Record 4 sets data [Latin square: Randomisation reduces error] as follows s1 s2 t t s1 s2 s2 t s1 s1 s2 t Plot mean of S1, S2 and T against dose. Calculate Log Potency ratio [ M ] = [ (T –S1) / (S2-S1) ] X log d d = s2/s1 Now calculate the conc of unknown as UK = s1/t X potency ratioX conc of unknown Error is calculated as error= calculate- actual/actual X 100

28. 3 point assay

29. 4 point assay [2 +2 dose assay] Procedure [ Eg Ach bioassay] Log dose response [LDR] curve plotted with varying conc. of std. Ach Select two std. doses s1& s2 from linear part of LDR Choose two test doses t1 & t2 Also s2/s1 = t2/t1 = 2

30. …Contd. Record 4 data sets with Randomisation s1 s2 t1 t2 s2 t1 t2 s1 t1 t2 s1 s2 t2 s1 s2 t1 Plot mean of S1, S2 and T1, T2 against dose. Calculate Log Potency ratio [M] = [ (T1 –S1 + T2 –S2) / (S2-S1 + T2-T1) ] X log d [d = dose ratio] d= s2/s1 Now calculate the concentration of unknown( unknown/ known = potency ratio Error is calculated as error= calculate- actual/actual X 100

31. Label s1= concentration of the standard s2= concentration of 2 nd standard dose S1 = response due to s1 S2 = response due to s2 t1= concentration of 1 st test dose t2= concentration of 2 nd test dose T1= response due to t1 T2= response due to t2

32. Contd… Microbiological assays Used for assaying antibiotics The test sample is compared with standard preparation for its inhibitory effect on particular organisms

33. Bioassay of Some Important Drugs Drugs Preparation Activity assessed Digitalis Cat blood pressure Guinea pig blood pressure Pigeon Fall in BP and death Stoppage of heart & death Emesis Adrenaline BP of spinal cat Isolated rabbit duodenum Isolated rat uterus Isolated caecum of fowl Rise in BP Inhibition of tone Inhibition of tone Inhibition of tone Noradrenaline BP of pithed cat Rise in BP Acetylcholine Isolated rectum Rectus of frog Rat ileum Isolated mouse heart Leech dorsal muscle Rat/cat BP Contractile effect Contractile effect Contractile effect Inhibition of contractions Contractile effect Fall in BP

34. Drugs Preparation Activity assessed Acetylcholine Isolated rectum Rectus of frog Rat ileum Isolated mouse heart Leech dorsal muscle Rat/cat BP Contractile effect Contractile effect Contractile effect Inhibition of contractions Contractile effect Fall in BP Histamine Isolated, atropinized terminal ileum of guinea pig Anaesthetized and atropinized cat Contractile effect Fall in blood pressure Adrenaline BP of spinal cat Isolated rabbit duodenum Isolated rat uterus Isolated caecum of fowl Rise in BP Inhibition of tone Inhibition of tone Inhibition of tone Bioassay of Some Important Drugs

35. Contd… Drugs Preparation Activity assesed Digitalis Cat blood pressure Guinea pig blood pressure Pigeon Fall in BP and death Stoppage of heart & death Emesis 5 HT Isolated atropinized rat uterus, Isolated terminal colon of rat Isolated fundus strip of rat stomach Perfused rabbit ear Contractile effect Contractile effect Contractile effect Constriction of blood vessels Heparin Whole blood of ox with thrombokinase extract and acetone dried ox brain Prolongation of blood clotting time Noradrenaline BP of pithed cat Rise in BP

36. Contd… Drugs Preparation Activity assessed Insulin Rabbit Mice Isolated rat diaphragm Rat's epididymal fat Lowering of blood-sugar Convulsions and/or death due to hypoglycaemia Increase in glycogen content Increased metabolism of glucose, indicated by increased in CO2 production Oxytocin Adult cockerel Isolated rat uterus Rabbits (female) Vasodepressor activity. Contractile effect Ejection of milk from mammary duct

37. Current status of bioassay: If one review the emphasis of bioassay in pharmacopeias published before 1980 as compared to those published recently, it will be clear that There are very few drugs which are now recommended to be assayed by biological methods. Most of the drugs which were assayed by biological methods are now being recommended to be assayed by chemical methods Newer drugs have been included for which bioassay recommended

38. Contd… Names of Drugs Assay method Recommended Acetylcholine Titrimetric method Histamine Titrimetric method Heparin Titrimetric method Thyroxine Titrimetric method Adrenaline UV absorbance Progesterone UV absorbance Digoxin UV absorbance Estradiol Chromatography Penicillin Chromatography Insulin HPLC

39. Contd… Recently newer techniques have come up which have replaced the bioassay techniques Eg chromatography for estradiol and penicilline & HPLC for insulin

40. To summarize Bioassay are defined as e stimation of the conc. / potency of a substance by measuring its biological response in living systems They are indicated when the active principle of the drug is unknown, chemical method is not available or is too complex or when the composition of the drug is variable They have been applied for variety of purpose like standardization of the drug or for research