Bioassay-part 1.pptx. pharmacology practical

BIOASSAY PART-1

1. BIOASSAY BY : Dr. SARITA SHARMA ASSOCIATE PROFESSOR DEPARTMENT OF PHARMACOLOGY MMCP, MMDU

2. OVERVIEW  BIOASSAY – Definition/Synonyms  PRINCIPLES OF BIOASSAY  INDICATIONS OF BIOASSAY  TYPES OF BIOASSAY  USES OF BIOASSAY 

3. 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. Bioassay is defined as the estimation of the potency of an active principle in a unit quantity of preparation. OR Detection and measurement of the concentration of the substance in a preparation using biological methods.

5. 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. 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. Cont d .. 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. Indications of bioassay (Application): 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. Cont d … 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. Bioassays, as compared to other methods of assay (e.g. chemical or physical assay) is very important. Because it is the only method of assay if Active principle of drug is unknown or cannot be isolated, e.g. insulin, posterior pituitary extract etc. Chemical method is either not available or if available, it is too complex and insensitive or requires higher dose e.g. insulin, acetylcholine. Chemical composition is not known, e.g. long acting thyroid stimulants. Chemical composition of drug differs but have the same pharmacological action and vice-versa, e.g. cardiac glycosides, catecholamines etc.

11. Bioassay can be performed on Intact an i m a ls Inv i vo Isolated tissues Specific cells Organisms Invi t ro

12. 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 ISOLATED TISSUE  Acetyl Choline – Frog Rectus Abdominus muscle  Histamine – Guinea Pig ileum  Adrenaline – Rat uterus  Oxytocin – Rat uterus oestrogen primed

13. Types of Bioassay QUANTAL ASSAY GRADED ASSAY

14. 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

15. 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 =

16. END POINT METHOD: The threshold dose producing a predetermined effect is measured Comparison between the results of standard and the test drug is done. e.g. bioassay of digitalis in cats. The cat is anaesthetized with chlorofom and its blood pressure is recorded. The drug is slowly infused into the animal.

17. The moment the heart stops beating and blood pressure falls to zero, the volume of fluid infused is noted down. Two series of such experiments-one using standard digitalis and the other using test preparation of digitalis is done . potency is calculated as follows: Threshold dose of the Std X Conc of Std Threshold dose of the Test Conc of Unknown =

18. 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 Test/unknown standard

19. 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.

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21. Bracketing or Direct Matching

22. ADVANTAGES  Simple & Faster  Amount of test drug available is small  Does not involve complicated calculations  Does not depend on DRC Disadvantages  less accurate, time consuming, troublesome  cannot get exact match of response  quantitative difference b/w test & std not obtained

23. 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

24. standard Test/unknown

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

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

27. 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

28. 34 t s 1 s2 s1 s2 t s 2 t s1 3 cycles 3 - point assay

29. 3-POINT ASSAY % R E S P O N S E 35 s1 T S2 t s2 LOG DOSE S1

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

31. 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

32. 38 4 - POINT ASSAY

33. 4-POINT ASSAY % R E S P O N S E 39 s1 T1 S2 s2 t1 LOG DOSE S1 T2 t2

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

35. 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

36. 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

37. 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 Determination of potency, ED50/LD50 of drugs  New drug development  Measure clinical effectiveness 45

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