Bioassay

Lecture includes definition of bioassay, Types of Assay and Bioassay , Indications, principles, advantages of bioassay. Example of a Bioassay with calculations. This lecture will be of help for postgraduate pharmacology students as well as undergraduates

1. Bioassay & DRC Dr Naser Ashraf Tadvi Associate Professor

2. Assay Analysis done to determine the presence of a substance & amount of that substance (Known or unknown) in biological fluids is known as Assay. Determines Activity and potency of drug

3. Types of Assays Biological Method (Bioassay) Physico -chemical methods Chromatography, spectrophotometry Radioimmunoassay Microbiological methods

4. Bioassay Measurement of the concentration or potency of a drug from magnitude of its main biological effect Main Pharmacological effect of drug Compared with Pharmacological effect of standard drug Potency ratios are compared quantitatively

5. Indications(Uses) of Bioassay Measure pharmacological activity of new/ chemically undefined substance Measure concentration of known substance in tissue extract or body fluid e.g Ach, histamine Measure ED50 / LD50 of a drug Biological standardization of natural drugs which cannot be obtained in chemically pure form e.g Vasopressin, oxytocin

6. Parameters recorded Blood pressure (Dogs and cats): Sympathomimetics Ach: contraction of rabbit duodenum or frog rectus abdominis Skeletal muscle relaxants: rabbit head drop method Histamine: Guinea pig ileum Serotonin on rat colon

7. Principles of Bioassay Experimental conditions should be constant Standard should be in pure form and known conc. Should be available Unknown and standard drug should have same mode of action and pharmacological effects Use a specified pharmacological technique like rectus muscle preparation for Ach System should be insensitive to other drugs

8. Characteristics of a good bioassay Sensitivity - ability to detect smallest concentration Specificity – the response which is being measured should specific. Reproducibility - same observations using different instrument and operator, over longer time periods. Stability – sensitivity of preparation should be constant. Availability – the particular tissue should be easily available

9. BIOASSAY Can be performed IN VIVO -On intact animal IN VITRO -Isolated tissues -Specific cells -Organisms in culture

10. Advantages of isolated tissue experiments over intact animal experiments Several preparations can be obtained from a single animal. Small amount of the test material is required. Cheap, less time consuming. Interference due to pharmacokinetic factors and compensatory reflexes is avoided.

11. Assembly for recording contraction of isolated tissue Organ Bath: Rudolph Magnus was first to design organ bath. Outer organ bath : made up of perplex with electrical thermostat and stirrer for uniform temperature. 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12. Assembly for recording contraction of isolated tissue Lever: Simple lever is used. Stylus

13. Magnification and Adjustment Magnification for Frog rectus is 5-10 Fast contracting- low Slow contracting- high B A A B

14. Role of Aeration / Aeration tube 1). Provides oxygen/ air to mounted tissue. 2). Allows uniform distribution of drug in organ bath through bubbles. 3). Areation tube helps in mounting tissue in organ bath. Aeration – 30-40 bubbles / min (air)

15. Assembly of instruments for bioassay Mariottes Bottle Sherringtons starling drum Simple writing lever Aeration tube Inner Organ bath Outer Organ bath Kymograph

16. 1 gm weight is added Writing lever Assembly of instruments for bioassay

17. Dissection and mounting the tissue Procedure: A pithed frog is laid on its dorsal surface in a dissection tray. The skin of the abdomen cut to expose the whole abdomen. The rectus muscle is identified, and the border of the muscle dissected out from adjacent abdominal wall. A thread is passed with the help of needle through the pubic attachment of muscle and tied ,muscle is cut. The upper end of muscle i.e , xiphisternum part also tied and cut. The lower end of the muscle is tied to aeration tube at ‘s’ bent.

18. Pithing and Dissection of Frog

19. Dissection and mounting the tissue Before mounting the tissue make sure the blood vessels are removed. Aeration tube with mounted tissue is placed in inner organ bath filled with PSS. The upper end of muscle is tied to lever with thread. A load of 1gm is attached to the other side of lever at same distance as that of tissue from the fulcrum. This helps the muscle to relax in between contraction. Tissue is aerated with air i.e 30-40 bubbles /min. and allowed to relax for 30- 45 min before recording responses.

20. Mounting the tissue

21. Acetyl choline Dilutions Stock solution- 1 mg/ml Acetylcholine(1000 μ g/ml) The dilutions are made as follows- 1 ml stock soln. + 9 ml distilled water= 1/10 dilution (100 μ g/ml) 1 ml 1/10 dilution + 9 ml distilled water= 1/100 dilution (10 μ g/ml) 1 ml 1/100dilution + 9 ml distilled water= 1/1000 dilution (1 μ g/ml)

22. Recording cycle Base line: 30 seconds(s) (start drum & allow to run freely) Contractions: 90 s (contact period of Ach with tissue) Relaxation: 3 minutes (tissue washed 2-3 times after 90 sec contact period) Total cycle is 5 mins Soon after contraction of 90 secs the weight of 1gm is lowered down to allow muscle to relax slowly, until lever comes to base line. The contractions are recorded with each dose of solution till ceiling effect is obtained i.e no further increase in height of contraction with subsequent doses.

23. Recording cycle After the ceiling effect is obtained with standard Ach solution, the procedure is repeated with the test solution(unknown strength of Ach) in different dilutions. The further experiment is done by Multiple point assay i.e. 2+1 assay Sensitivity of Rectus muscle can be increased by adding Physostigmine (0.5 μ g/ml)/ Alcohol to perfusion fluid

24. Types of Bioassay A. Quantal (Direct end point) Assay B. Graded Assay -Bracketing assay -Matching assay -Interpolation assay -Multiple point assay

25. 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. Ex: Digitalis induced cardiac arrest in cats, insulin induced hypoglycemic convulsions in mice.

26. Quantal response (All or none assay)

27. Graded response assay

28. Multiple Point Assay Bracketing and Matching assay methods are not ideal because lack of accuracy, sensitivity So , to minimize those errors multiple point assays preferred (3,4 or 6 point assays)

29. 0.2 100 25.6 12.8 6.4 3.2 1.6 51.2 0.8 0.4 10 U 400 200 40 U 20 U 80 U 16 U 32 U 64 U S2 S1 S1 S1 S1 S2 S2 S2 t t t t 2+1 Bioassay of Ach on Isolated Frog Rectus Muscle S2 S1 T

30. 6 Dose response curve

31. Test 1/10 10 U 80 U 40 U 20 U Test SS 64 U 32 U 16 U Dose response curve of test solution in graded doses

32. Latin square S1 S2 t t S1 S2 S2 S1 t X

33. Calculations Response Height of contraction (mm) Mean height (mm) Dose (ml) 1 2 3 S 1 34 32 37 35.2 0.1 S 2 67 65 80 72.7 0.2 T 42 42 43 40.7 0.32 Dose ratio(d) = = = 2 S 1 S 2 0.2 0.1

34. From data obtained log potency ratio (M) is calculated from the formula: M = × log d M = × log 2 M = × 0.301 = 0.047 Calculations T – S1 S2 – S1 41 - 35 73 - 35 6 38

35. From M ,the strength of the unknown can be calculated – Strength of = × antilog of M test solution = × 1.114 = 0.348125 mg/ml = 348 μ g/ml The given concentration was 300 μ g/ml , so the error is 16% Percentage error(%)= × 100 Calculations S1 t 0.1 0.32 ACT ACT - OCT

36. Calculation DRUG DOSE ( μg) RESPONSE (mm) LOG DOSE 2 0.30103 4 0.60206 8 0.90309 16 4 1.20412 32 8 1.50515 64 24 1.80618 128 30 2.10721 256 40 2.40824 512 42 2.70927 1024 45 3.0103 2048 45 3.31133

37. Dose Response Curve DOSE ( μg) RESPONSE (mm)

38. Calculation of ED 50

39. LDR CURVES OF 2 DRUGS WITH DIFFERENT POTENCY

40. EFFICACY & POTENCY

41. FREQUENCY DISTRIBUTION CURVE

42. Therapeutic Index ED 50 LD 50

43. Certain Safety Factor

44. Therapeutic window

45. Matching/ Bracketing Assay

46. Matching/ Bracketing Assay

47. Matching/Bracketing Assay 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

48. Interpolation method

49. Interpolation method

50. Thank You