Bioassay and it's Methods & Applications

Hello here is all points covered related to the Pharmacology III in that bioassay syllabus point

1. Pharmacology-II Topic- Bioassay -Prepared By- Mr. Sanket Ravindra Ahirrao ( T.Y.B.Pharm ) -Guidance By- Prof. Zeeshan Munaf Nathani {M. Pharm- Pharmacology} (Department- Pharmacology) OBVS’s Prof. Ravindra Nikam College of Pharmacy, Gondur , Dhule (424002)

2. Bioassay: Introduction Definition:- Estimation of the conc. Or potency of a substance by measuring its biological response in living systems. Principle of Bioassay:- Compare the biological effect produced by the test substance with that of standard preparation and find out how much test substance is required to produce same biological effect as produced by the standard.

3. Types Of Bioassay:- There are mainly Two types of Bioassay and their Subtypes as Mentioned Below- Graded Assay- Matching Bracketing Interpolation Multiple Point 2) Quantal Assay- Direct End point Assay (DEPA) LD⁵⁰ Determination

4. Graded response Assay – response is proportional to the dose and response may lie between no response and the maximum response. a) Matching Bioassay- In cases, where sample size is very small. Test response is matched against standard response by Trial & Error method. Potency ratio of Test & standard are calculated. Concentration of test sample are calculated from concentration of standard. Example: Bioassay of Histamine,Adrenaline , Acetylcholine.
Demerit: DRC is not recorded. Hence, Precision & Reliability of this method is poor.

5. b) Bracketing Assay- 1)It involves bracketing of test response between one higher & one lower response of a standard drug.
2) Interpolation of the bracket response on Dose-axis gives concentration of test sample.
3)It’s a simple method.
4)It requires very small amounts of sample.

6. C) Interpolation Method- 1)Bioassays are conducted by determining the amount of preparation of unknown potency required to produce a definite effect on suitable test animals/organs/Tissue under standard conditions. 2) This effect is compared with that of a standard. 3)Thus the amount of the test substance required to produce the same biological effect as a given quantity the unit of a standard preparation is compared and the potency of the unknown is expressed as a % of that of the standard by employing a simple formula.

7. d) Multiple-point Bioassay - i ) Three-point assay: 1) 2 standard & 1 test response taken in to consideration. 2) 1st-Log DRC for standard drug is plotted. 3) 2 doses S1 & S2 whose response lies within linear portion of curve are then selected. 4) The concentrations of these doses should be in simple ratio. 5) Dose of unknown test drug preparation that produces a response in between S1 & S2 is obtained. 6) 3 doses (t,s1,s2) are bioassayed using L ycergic Acid Diethylamide(LSD) . 7) Mean responses for each set of doses (s1,s2,t) are plotted to obtain log DRC.

8. ii) Four-point assay: 1) 2 standard responses; 2 test responses are taken in to consideration.
2) Log DRC of standard & test drug are plotted initially.
3) Two standard doses are selected in a way that their response lie in between linear portion of log-DRC & ratio between the doses is 1:2.
4)Test responses are selected such that they lie on linear portion of log DRC.
5) They should be selected such that responses at lower & higher doses of both standard & test preparations are similar.
6) Ratios of test & standard doses.i.e ; t2/t1 & s2/s1 should be simple & same.

9. 2) Quantal Assay- Direct End Point Assay (DEPA):- 1) Doses of the standard and test preparations are sufficient to
produce a specified response and can be directly measured.
2) The principle of direct assay is to measure direct response of
dose of standard and test preparation.
3) Mainly two experimental designs are followed, one is crossover, and another is parallel group or completely randomized design.
• Example: Assay of digitalis in cat.

10. 2) LD⁵⁰ Determination- 1)The unknown is compared with the standard with respect to potency which produces the quantal affect, i.e. Changes is easily recognized sign or often death.
2)In a quantal assay there is use of dose response relationship, however assay are more closely related to direct assay.

•Example: Determination of LD Assay of insulin by mice convulsion method

11. Applications of Bioassay- For screening pharmacological activity of new/chemically undefined substances. B) To measure potency/concentration of substance in tissue/body fluids. C) To estimate LD50 &ED50 of drug. D) For biological standardization of drugs of natural origin which cannot be derived in pure form. • Example: Insulin , Heparin,Oxytocin .

12. For the following categories Bioassay is to be indicated:

✓ For drugs of unknown chemical composition,

✓ Drugs for which chemical assay is complex. Example: Adrenaline & Histamine.

✓ Drugs which have same pharmacological activity but different chemical composition. • Example: Digitalis glycosides obtained from different plant sources.

✓ Drugs that do not have an adequate chemical assay Example: Insulin

13. Bioassay of Insulin:- Bioassay of Insulin is performed by a Rabbit Method- • Preparation of test sample solution: The solution of the test sample is prepared in the same way as the standard solution described above. # Rabbit Method: • Selection of rabbits: They should be healthy, weighing about 1800-3000 gms . They should then be maintained on uniform diet but are fasted for 18 hrs. Before assay. Water is withdrawn during the experiment.

14. • Standard and Sample Dilutions: These are freshly prepared by diluting with normal NaCl solution so as to contain 1 unit/ml and 2 units/ml. • Doses: The dose which can produce suitable fall in blood sugar level is calculated for the standard. • Principle: The potency of a test sample is estimated by comparing the hypoglycemic effect of the sample with that of the standard preparation of insulin. Any other suitable method can also be used. •Experimental Procedure: -Animals are divided into 4 groups of 3 rabbits each.
-The rabbits are then put into an animal holder. -They should be handled with care to avoid excitement.

15. = First part of the Test: • A sample of blood is taken from the marginal car vein of each rabbit. Presence of reducing sugar is estimated per 100 ml of blood by a suitable chemical method. This concentration is called ‘Initial Blood Sugar Level • The four groups of rabbits are then given sc. Injections of insulin as follows: -3 Standard Dilution(1) -3 Standard Dilution (II)
-3 Test sample dilution(I)
-3 Test sample dilution(II) • From each rabbit, a sample of blood is withdrawn up to 5 hrs. At the interval of 1 hr. Cach . Blood sugar is determined again. This is known as ‘Final Blood Sugar Level’.

16. = Second part of the test (Cross over test): The same animals are used for the second part. The experiment can be carried out after one week. Again they are fasted and initial blood sugar is determined. The grouping is reversed, that is to say, those animals which received the standard are given the test and those which received the test are now given the standard. Those animals which received the less dose of the standard are given the higher dose of the test sample and vice-versa. This test is known as ‘Twin Cross Over Test’ Mean percentage decrease in blood sugar of the first and second part is calculated.

17. Bioassay of Oxytocin:- OXYTOIN ( IP’07) 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: consisting of freeze-dried synthetic oxytocin peptide with human albumin and citric acid (supplied in ampoules containing 12.5 Units).

18. Method: • By contraction of the rat uterus • Inject 100 mg of oestradiol benzoate intramuscularly into a female rat weighing 120 to 200 g 18 to 24 hours before the assay. • Kill the rat and suspend one hom of the uterus in a bath containing a solution of the following composition.

19. • Maintain the bath at 32” Cat which spontaneous contractions of the uterus are abolished and the preparation maintains its sensitivity.
• Oxygenate the solution with a mixture of 95% of oxygen and 5% of
carbon dioxide
• Record the contractions of the muscle using a suitable instrument giving a linear response
• Record the contractions produced by the addition of two doses ofthe Standard Preparation suitably diluted with the above solution

20. • The doses should be such as to produce clearly discriminated contraction.

• The required doses normally lie between 10 and 50 micro Units per ml of bath liquid.

• The doses should be added at regular intervals of 3 to 5 minutes depending upon the rate of recovery of the muscle.

• Dilute test preparation so as to standard produce same response as that of Standard.

21. • The ratio between the two doses of the preparation being examined should be the same as that of the Standard Preparation and this ratio should be kept constant throughout the assay.

• The two doses of Standard Preparation and the preparation being examined should be given according to a randomized block or a Latin
square design and at least six responses to each should be recorded.

• Calculate the result of the assay by standard statistical methods

22. Thank You For Your Attention!!!