Bioassay

pharmacology -bio assay

1. BIOASSAY Dr Manish Mohan JR Pharmacology

2. Contents Introduction Objectives of assay Characteristics of good assay Errors in bioassay Methodology Organ bath Physiological solution Different types of assay

3. ASSAY “Estimation of the amount or activity of an active principle in a unit quantity of preparation”

4. Types of assay 1.Physiochemical - gas chromatography - spectrophotometry -fluorimetry - mass spectrometry 2. Biologically mediated/bioassay 3.Immunological assay – RIA, ELISA 4.Microbiological assay

5. DEFINITION “ Bioassay is defined as comparative assessment of relative potency of a test compound (T) to a standard compound (S) on any living animal or biological tissue ”

6. INTRODUCTION Standardization of diphtheria antitoxin -Paul Ehrlich Basic step towards the drug discovery Determine the effect of any natural source of unknown substances Assessment of unknown substance on any living tissue

7. A bioassay experiment - Quantal - Quantitative Substances Used in Bioassay - Derived from plants and animal sources

8. Objective or Purpose of Bioassay Identification of various compounds Quantify the screening procedure Commercial production of drugs like antibiotics

9. Cont …….

10. Characteristics of good assay method High sensitivity High specificity Repeatability Reproducibility Accuracy Stability

11. PRINCIPLES OF BIOASSAY

12. ERROR IN BIOASSAY Biological variation Methodological error

13. Reason and its correction for biological variation Downregulation of receptors (repeated washing of tissue; avoid over washing) Loss of tissue sensitivity (change the tissue) • Animal use of different species, sex, age, weight and health status (must use same species) Laboratory condition may be variable (must be kept constant) Housing and handling of animals (must be handled by a qualified experienced staff)

14. Reasons for Methodological error and its correction Lack of standardization of procedure (standardize your procedure beforehand) Set-up of apparatus (Check every instrument used in the experiment for their proper working) Tissue isolation/extraction and preparation for the experiment (minimize handling and excess cleaning of the tissue while mounting in the organ bath)

15. Cont ….. Preparation of physiological salt solution (PSS) and maintenance of its pH (Follow the standard procedure to prepare and maintain the pH of PSS) Drug preparation or it dilution (while making serial dilution of the drug, mix it slowly; Avoid the vigorous mixing).

16. Bioassay may be performed on 16

17. WHOLE ANIMALS Nor Adrenaline – Spinal Cat Cardiac Glycosides – Guinea Pig Insulin – Mice Estrogens – Ovariectomized Female Rat MICRO ORGANISMS Vit B 12 – E uglena gracilis 17

18. ISOLATED TISSUE Acetyl Choline – Frog Rectus Abdominus muscle Histamine – Guinea Pig ileum Adrenaline – Rat uterus Oxytocin – Rat uterus estrogen primed DISPERSED CELLS Plasma LH estimation by stimulation of testosterone synthesis - on isolated Leydig cells   18

19. METHODOLOGY

20. ORGAN BATH “ Traditional experimental set-up that is commonly used to investigate the physiology and pharmacology of in vitro tissue preparations” Perfused tissues can be maintained for several hours in a temperature controlled organ bath

21. Essential Parts of Organ Bath which is Required to Record the Response of a Tissue/Muscle Sherrington rotating drum (standard speed of the drum is 1 revolution/96 min.) Kymogram is the paper used to record the response Outer bath/Outer water jacket Inner organ bath

22. Cont.…. Tissue holder and oxygen supply Fulcrum Heating iron coil and thermostat Fixing of the recording (shellac and colophony saturated in the alcohol)

23. METHODOLOGY

24. Physiological Salt Solution Also called as PSS / Ringer solution Maintains tissue outside the animal body Select PSS in which tissue last longer Prepare the solution with the help of distilled or double distilled or deionized water Prepare fresh solution

25. PHYSIOLOGICAL SALT SOLUTION (PSS)

26. Role of Each Ingredient Sodium (Na+): maintain the osmolarity Potassium (K+): maintaining rate and rhythm. Calcium chloride (CaCl2): excitation Magnesium chloride (MgCl2): reduce the spontaneous activity of tissue

27. Amphibian tissue - Frog-Ringer solution heart muscle preparation- Ringer-Locke Tyrode - mammalian smooth muscle Krebs, De Jalon and McEwen - mammalian isolated organ

28. PHYSIOLOGICAL SALT SOLUTION (PSS) Tyrode may be used for experiment with non-innervated muscle while Krebs is used for nerve muscle preparation PH- 7.3-7.4

29. LEVER “Load force × length of load arm = Effort force × length of effort arm.” Effort arm Load arm Fulcrum

30. Different types of levers, commonly used in experimental pharmacology Simple lever Frontal writing lever

31. Gimbal lever Sprung lever Auxotonic lever Starling heart lever

32. Magnification

33. TYPE OF TISSUE According to tissue/muscle obtained: – Smooth muscle preparation, e.g.: Uterus, tracheal smooth muscle, vas deferens, aorta, ileum, ascending or descending colon, etc. – Skeletal muscle, e.g.: Rectus abdominus muscle, etc. – Cardiac muscle, e.g.: Frog heart etc.

34. 2. According to response obtained by tissue/ muscle - Slow contracting tissue/muscle e.g. Frog rectus abdominus muscle, stomach fundus, guinea pig tracheal smooth muscle – Fast contracting tissue/muscle e.g. Ileum, uterus, ascending and descending colon

35. CLASSIFICATION OF BIOASSAY 1. Direct end point assay (DEPA) 2. Quantal assay (all or none assay) 3. Graded assay a. Bracketing assay b. Matching assay c. Interpolation assay d. Multiple point assay (3-point, 4-point, 6-point and 8-point)

36. 1. End Point Method Threshold dose producing a positive effect is measured e.g. bioassay of digitalis in cats The threshold dose of standard = Total period of infusion × Rate of drug administration

37. Concentration of test = TDS X CSD TDT TDS = Threshold dose of standard TDT = Threshold dose of test CSD = Concentration of standard drug

38. Disadvantages Only toxicity study or high dose study is possible Dose ranging study cannot be done.

39. 2 . Graded response assays Response is proportional to the dose Response may lie between no response and the maximum response

40. Bracketing assay Test sample volume is too small Single /few responses – test drug This response is bracketed b/w 2 responses of the standard drug Potency – conc. Standard drug / dose response curve

41. Matching assay Compare both test and standard – trial and error method Response is matched b/w test and standard drug Require very small volume of test drug It require most sensitive tissue

42. Disadvantage Purely subjective Experimental error cannot be determined Require Most sensitive tissue

43. Interpolation Assay Concentration of unknown is interpolated from the DRC graph

44. Limitations of above mentioned assays Lack of sensitivity Lack of accuracy Methodological errors such as tissue sensitivity error Variable temperature error Dilution error

45. 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

46. 3 POINT METHOD - 2 doses of std+1 dose of test

47. Calculation S1 and S2 = Length of standard dose response T = Length of test dose response s1 and s2 = Doses which produces responses S1 and S2

48. CONT…… Percentage error (%) = ACT – OCT X 100 ACT where, ACT = Actual concentration of test OCT = Observed concentration of test

49. 4 point method - 2 doses of std+2 doses of test

50. 38 4 - POINT ASSAY

51. 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

52. 6-point and 8-point Assay Time consuming lengthy procedure Reduced error Greater specificity

53. APPLICATIONS OF BIOASSAY Estimate potency of natural drug. 2. Standardization of drugs of natural origin (plant and animal origin) 3. Screening of new compound for biological activity 4. Estimation of biologically active substance like histamine, acetylcholine, adrenaline.

54. Thank you