Bioassay

1. BIOASSAY Dr. RENJU.S.RAVI

2. OVERVIEW BIOASSAY – Definition/Synonyms PRINCIPLES OF BIOASSAY INDICATIONS OF BIOASSAY TYPES OF BIOASSAY USES OF BIOASSAY DRAWBACKS BIOASSAY IN HUMANS

3. What is ASSAY Amount or activity of an active principle in unit quantity of preparation Types : 3 Photometry Chromatography RIA ELISA

4. Bioassay Potency or concentration of an active principle in unit quantity of preparation by measuring its biological response on living tissues Introduced by Paul Ehrlich - biostandardization of Diphtheria antitoxin 4

5. Sy nonyms 5

6. Principles of bioassay To compare the test substance with the International Standard preparation of the same To find out how much test substance is required to produce the same biological effect, as produced by the standard 6

7. Contd.. Activity assayed should be the activity of interest Standard & test sample - similar pharmacological effects & mode of action Both should be compared for their established pharmacological effect using specified technique Ex: *Ach – contractile response on frog rectus *Histamine – contractile response on guinea pig ileum 7

8. Contd .. Problem of biological variation must be minimized Experimental conditions - kept constant Animals - same species, sex and weight Number of animals - large enough to minimize error (individual variation) Isolated preparations - sensitive 8

9. Indications of bioassay No chemical method has been developed Chemical assay is too complex /not sensitive enough to measure (ex: insulin, Ach) To measure the pharmacological activity of new or chemically undefined substances For biological standardization of drugs obtained from natural sources as these cannot be obtained in pure form. Eg : Oxytocin,Vasopressin,Insulin,Heparin .. 9

10. Contd … To compare the strength of a drug obtained from various sources due to different compositions ( Eg:Cardiac glycosides ) Chemicals with similar structure, but different biological activity Chemical structure of the active principle is unknown Chemical structure known; cannot be actively purified. Eg : Peptide hormones 10

11. Characteristics of a good assay method Sensitivity Specificity Repeatability Reproducibility Precision Accuracy Stability – tissue has to stay “bioassay-fit 11

12. Bioassay can be performed on 12

13. WHOLE ANIMALS Nor Adrenaline – Spinal Cat Cardiac Glycosides – Guinea Pig Insulin – Mice Estrogens – Ovariectamised Female Rat MICRO ORGANISMS Vit B 12 – E uglena gracilis Tetracycline - Bacillus pumilus 13

14. ISOLATED TISSUE Acetyl Choline – Frog Rectus Abdominus muscle Histamine – Guinea Pig ileum Adrenaline – Rat uterus Oxytocin – Rat uterus oestrogen primed DISPERSED CELLS Plasma LH estimation by stimulation of testosterone synthesis - on isolated Leydig cells   14

15. Types of Bioassay QUANTAL ASSAY GRADED ASSAY INDIRECT ASSAY DIRECT ASSAY

16. Indirect assay Potency of test is estimated by comparing the Log Dose-Response curve with a similar curve of the standard Eg : Ergot preparation in White Leg-Horn Cock DIRECT ASSAY Dose of test to elicit ED50 or LD50 is measured Direct comparison on isolated tissue:- Interpolation Method, Matching, Four Point Assay Direct assay on living animals:- Digitalis bioassays in guinea pigs- Cardiac arrest 16

17. Quantal assay Quantal response - the response is in the form of "all or none", i.e. either no response or maximum response Drugs producing quantal effect can be bioassayed by end point method 17

18. The threshold dose producing a predetermined effect is measured Comparison between the results of standard and the test E.g : Bioassay of digitalis in cats, Insulin induced hypoglycemic convulsions in rat Threshold dose of the Std X Conc of Std Threshold dose of the Test Conc of Unknown =

19. Graded assay Graded response - response is proportional to the dose and response may lie between no response and the maximum response . Types : Bracketing /direct matching Interpolation Multiple point assays Three point assay Four point assay Six point assay Cumulative dose response 19

20. Graded DRC 20 standard Test/unknown

21. 21 DRC & Log DRC 30% 70% Sigmoid curve Wide range of doses can plot Rectangular hyperbola Potency Efficacy Slope of curve

22. 22

23. Bracketing or Direct Matching A constant dose of the standard is bracketed by varying dose of test sample until an exact matching between the response of std & that of the sample is achieved Strength of unknowm /test drug can be found by simple interpolation of bracketed response. 23

24. 24

25. Bracketing or Direct Matching 25

26. ADVANTAGES Simple & Faster Amount of test drug available is small Does not involve complicated calculations Does not depend on DRC 26

27. Disadvantages less accurate,time consuming, troublesome cannot get exact match of response quantitative difference b/w test & std not obtained

28. Interpolation assay A log dose-response curve is plotted with the standard on a simple graph paper or Semi-log paper The concentration of the test is then read from the graph 28

29. 29 standard Test/unknown

30. Interpolation 100 50 0 x x 1 standard % R E S P O N S E LOG DOSE

31. Advantages Sensitivity of tissue is 1 st determined by prior plotting of a conc -response curve with known agonist Dose can be plotted even if it varies over thousand fold range Error is normally distributed

32. Disadvantages Sensitivity of tissue changes with time Timing of doses not taken into account Variation in mode of application of drugs

33. Multiple point assays Responses are repeated several times and the mean of each is taken Chances of error are minimized 3 point method - 2 doses of std+1 dose of test 4 point method - 2 doses of std+2 doses of test 6 point method - 3 doses of std+3 doses of test Latin square method of randomization to avoid any bias 33

34. 34 t s1 s2 s1 s2 t s2 t s1 3 cycles 3 - point assay

35. 3-POINT ASSAY % R E S P O N S E LOG DOSE 35 s1 T t S2 s2 S1

36. Latin square design s 1 s 2 t t s 1 s 2 s 2 s 1 t t s 2 s 1

37. CALCULATION Mean responses of these 3 sets plotted Log potency ratio (M) = (T-S1÷ S2-S1)× log d where, d – dose ratio = s2/s1 Strength of unknown = s1/t × antilog of M 37

38. 38 4 - point assay

39. 4-POINT ASSAY % R E S P O N S E LOG DOSE 39 s1 T1 t1 S2 s2 S1 T2 t2

40. Latin square design s 2 t 1 t 2 s 1 t 1 t 2 s 1 s 2 t 2 s 1 s 2 t 1 s 1 s 2 t 1 t 2

41. Calculation Mean responses of 4 sets plotted Log potency ratio (M) (T2-S2)+(T1-S1) × Log d (S2-S1)+(T2-T1) where, d-dose ratio = s2/s1 Strength of unknown = s1/t1 × antilog of M 41

42. Six point assay 3+3 dose assay 3 conc each of std & test drug are used 6 sets of experiments using 6 doses in each set More time consuming,lesser in use Reliability is excellent

43. Cumulative Dose Response Curve Increase conc of drug in bath fluid step by step without washing out the preceeding doses Continue till supramaximal effect is seen Dose response curve is plotted

44. Cumulative dose response

45. Uses of Bioassay to measure the pharmacological activity of new/ chemically undefined substances to investigate the function of endogenous mediators to measure drug toxicity and unwanted effects to measure the conc of drugs and other active substances in the blood or other body fluids 45

46. Determination of potency, ED50/LD50 of drugs New drug development Measure clinical effectiveness 46

47. Drawbacks Biological variation Troublesome Time consuming Expensive Less accurate than physico -chemical methods 47

48. HUMAN TISSUE BIOASSAY • Veins [ surgery on varicose veins ] • Tissues like larger blood vessels obtained during amputation • Organs removed during transplantation/ tumor surgeries/procedures requiring resection • Tissues collected Postmortem 48

49. Conclusion Successful tool in estimation & discovery of biologically active substances Sensitivity & Specificity – important tool in pharmacology