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  1. 1. Integral university SeminarOn BIOASSAY OF DRUGS By: underguidance of: Shaikh ZohraMeena Dr. Anuradha Mishra Dr. Badruddin M.PHARMA (Pharmacology) 1st year
  2. 2.  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.
  3. 3.  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 1. Active principle of drug is unknown or cannot be isolated, e.g. insulin, posterior pituitary extract etc. 2. Chemical method is either not available or if available, it is too complex and insensitive or requires higher dose e.g. insulin, acetylcholine. 3. Chemical composition is not known, e.g. long acting thyroid stimulants. 4. Chemical composition of drug differs but have the same pharmacological action and vice-versa, e.g. cardiac glycosides, catecholamines etc.
  4. 4.  The basic principle of bioassay is to compare the test substance with the International Standard preparation of the same and to find out how much test substance is required to produce the same biological effect, as produced by the standard.
  5. 5.  Mainly two types are there. Graded Quantal
  6. 6.  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.
  7. 7.  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
  8. 8. 1. 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 chloralose and its blood pressure is recorded. The drug is slowly infused into the animal.
  9. 9. 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 Standard Conc. of Unknown = X Conc. of Std. Threshold dose of the Test
  10. 10. 2. Matching and bracketing method 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. Initially, two responses of the standard are taken. The doses are adjusted such that one is giving response of approximately 20% and other 70% of the maximum. The response of unknown which lies between two responses of standard dose is taken.
  11. 11. The panel is repeated by increasing or decreasing the dose s of standard till all three equal responses are obtained. The dose of test sample is kept constant. At the end, a response of the double dose of the standard and test which match each other are taken. These should give equal responses. Concentration of the test sample can be determined as follows: Dose of the Standard Conc. of Unknown = X Conc. of Std. Dose of the Test
  12. 12. Example: Bioassay of Histamine on guinea pig ileum.
  13. 13. 3. Graphical method This method is based on the assumption of the dose-response relationship. Log-dose-response curve is plotted and the dose of standard producing the same response as produced by the test sample is directly read from the graph. In simpler design, 5-6 responses of the graded doses of the standard are taken and then two equiactive responses of the test sample are taken. The height of contraction is measured and plotted against the log- dose.
  14. 14. The characteristic of log-dose response curve is that it is linear in the middle (20-80%). Thus, the comparison should be done within this range only. In other words, the response of test sample must lie within this range.
  15. 15. Dose Log dose Response (height) (cm) % response 0.1 -1 0.9 25.71 0.1 -1 0.9 25.71 0.2 -0.69 1.5 42.85 0.4 -0.39 2 57.14 0.6 -0.22 2.8 80 0.8 -0.09 3.5 100 1 0 2.3 65.71 0.1 -1 0.7 20 0.3 -0.52 1.3 37.14 Observations and calculation For standard For test
  16. 16. Maximum response (3.5) is considered 100% response. For 0.9, % response = 0.9 x 100 = 25.71 3.5 100 50 0 % R E S P O N S E Log dose
  17. 17.  Other methods which are based on the dose-response relationship include 3 point, 4 point and 6 point methods.  In these 3 methods, the responses are repeated several times and the mean of each is taken.  Thus, chances of error are minimized in these methods.  In 3 point assay method 2 doses of the standard and one dose of the test are used.  In 4 point method 2 doses of standard and 2 doses of the test are used. In 6 point method 3 doses of standard and 3 doses of the test are used.  Similarly one can design 8 point method also.
  18. 18.  The sequence of responses is followed as per the Latin square method of randomization in order to avoid any bias.  The mean responses are calculated and plotted against log-dose and amount of standard producing the same response as produced by the test is determined graphically as well as mathematically: n1 T - S1 n2 Cs Conc. of Unknown = t X antilog S2 - S1 X log n1 n1 = Lower Standard dose n2 = Higher Standard dose t = Test dose S1 = Response of n1 S2 = Response of n2 T = Response of test (t) Cs = Concentration of standard
  19. 19. Bioassay of histamine by three point method.
  20. 20. Similarly, in 4 point method, amount of standard producing the same response as produced by the test can be determined by graphical method. It is determined mathematically as follows: n1 (S1 + S2) – (T1 + T2) n2 Conc. Of unk= t1 X antilog (S2 + T2) - (S1 + T1) X log n1 t1 = lower dose of test t2 = higher dose of test T1 = response of t1 T2 = response of t2.
  21. 21.  Commonly used method for the bioassay of antagonist is simple graphical method.  The responses are determined in the form of the % inhibition of the fixed dose of agonist.  These are then plotted against the log dose of the antagonist and the concentration of unknown is determined by finding out the amount of standard producing the same effect as produced by the test.
  22. 22.  In this 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 responses of agonist are repeated every 10 mins till recovery is obtained.  The higher dose of standard antagonist is added and responses are taken as before.  Three to four doses of the standard antagonist are used and then one to two doses of test sample of the antagonist is used similarly.
  23. 23.  The percentage inhibition is calculated, plotted against log dose of antagonist and the concentration of unknown is determined as usual.
  24. 24. D- tubocurarine DigitalisOxytocin
  25. 25. 1. Rabbit Head-drop Method :  Principle: d-Tubocararine hydrochloride is injected into the marginal vein of a rabbit’s ear till the rabbit’s neck muscles are relaxed such that the animal cannot hold its head up. The total amount of test sample required to produce the endpoint is compared with the total amount of the standard sample required to produce similar endpoint.  Selection of Rabbits: Rabbits weighing 2 kg. are used. Animals should be free from disease, obtained from a healthy colony and should be accustomed with the experimental procedure.
  26. 26. Experimental Procedure: o Rabbit is placed in a holder with its head protruding outside. o The head should be freely movable. o Minimum 8 rabbits are used. Rabbit head drop method for the bioassay of d-tubocurarine. (i) : i.v. inj. of d-tubocurarine. (ii) : Head drop after injection.
  27. 27. o They are divided into two groups each containing 4 rabbits. o First group will receive standard sample and the second group will receive the sample under test. o d-Tubocurarine solution is injected at a constant speed by infusion apparatus through the marginal vein. o Injection should be given at a rate of 0.4 ml/min and should take about 10 min. Dose 0.012% w/v in saline.
  28. 28. o Infusion is continued till the rabbit will not be in a position to hold its head erect or there will be no response by focusing light on the eyes. o Rabbits recover immediately from the effect of curarization. o During the experiment there is a possibility of respiratory embarrassment which is treated by injecting neostigmine, methyl sulphate (0.05 mg.) and atropine sulphate immediately through the marginal ear vein.
  29. 29. o Cross-over test is carried out to minimize biological error due to animal variation. o Those rabbits which received the standard sample on the first day will be given test sample on the second day of experiment and vice versa. o Mean dose which produces head drop of the test sample is compared with the mean dose of standard preparation.
  30. 30.  Principle: Potency of the test sample is compared with that of the standard preparation by determining the action on the cardiac muscle.  Standard Preparation and Units: The standard preparation is a mixture of dried and powdered digitalis leaves (1 unit = 76 mg.)  Preparation of Extracts: Exact amount of the powder is extracted with dehydrated alcohol in a continuous extraction apparatus for six hours. The final extract should contain 10 ml. (5 ml. alcohol + 5 ml. water) per 10 g. of digitalis powder. It should be stored in between 5 oC and –5 oC.
  31. 31. o Minimum 6 pigeons are used for testing each sample. o The weight of the heaviest pigeon should not exceed twice the weight of the lightest pigeon. o Food is withheld 16-28 hours before the experiment. o Pigeons are divided on the basis of their sex, weight and breed, into two groups.
  32. 32. o They are anaesthetized with anesthetic ether. o One side of the wing is dissected and the alar vein is cannulated by means of a venous cannula. Dilutions are made with normal saline. o The test sample and standard sample is infused through cannula.
  33. 33. o In pigeons, stoppage of heart is associated with a characteristic vomiting response called ‘emesis’. o The milk from the crop sac of pigeons is being ejected out. This may be taken as the end point response of digitalis. o The lethal dose per kg. of body weight is determined for each pigeon. o The potency of the test sample is determined by dividing the mean lethal dose of standard by the mean lethal dose of the test sample.
  34. 34.  Principle: The potency of oxytocin is determined by comparing its activity with that of the Standard Preparation of oxytocin under the conditions of a suitable method of assay.  Standard Preparation: The Standard Preparation is the 4th International Standard for Oxytocin, established in 1978, consisting of freeze-dried synthetic oxytocin peptide with human albumin and citric acid (supplied in ampoules containing 12.5 Units)
  35. 35. o Anaesthetize a young healthy adult cockerel weighing 1.2 to 2.3 kg with an anesthetic that will maintain a prolonged and constant high blood pressure. o Expose the gluteus primus muscle in one thigh and cut and retract it to reveal the popliteal artery and crural vein. o Cannulate the popliteal artery and record the blood pressure. o Cannulate the crural or brachial vein.
  36. 36. o Prepare standard solution with saline. Inject 0.1-0.5 ml. o Inject 2 doses of standard solution into cannulated vein and record blood pressure. o Dose should cause decrease in B.P. o Interval between 2 injections is 3-10 mins or depends on rate at which B.P. comes to normal. o Dilute test sample with saline same as standard one.
  37. 37. o Ratio between standard and test should be equal. o If animal become insensitive due to repetitive doses so another animal is taken. o Measure all responses and result is calculated by standard statistical method.