Bioassay ,its types for theory & practical

BIOASSAY ,TYPES , BIOASSAY GRAPH

1. BIOASSAYS PRESENTED BY HEENA PARVEEN , PHARMACOLOGY DEPT. ASSISTANT PROFESSOR.

2. BIOASSAY BIO= LIFE ; ASSAY= TO TEST The technique involved in estimation of potency of an active constituent in a unit quantity of preparation from the magnitude of its main biological effect is called Bioassay/Biological standardization. Aim : To determine the amount of active ingredient present in a preparation . Example : Parasympathomimetic action of unknown drug is determined comparing its contractility with the standard solution of Acetylcholine {parasympathomimetic}. FACTORS INFLUENCING ACCURACY & RELIABILITY ARE: Selection of Animals, Selection of Test procedure, Details of test procedure, Trained staff performing bioassay.

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

4. Principles of Bioassay These principles of Bioassay is constituted by BURN & DALE . Basic Principle underlying bioassay __ To compare the test substance with the international standard preparation of same & to estimate how much test substance is needed to produce same biological effect as produced by standard. Other principles as follows are: Biological response produced by active principle to be measured in bioassay should be same in all species. The Method selected should be Reliable, Sensitive & Reproducible. Animals of same species, sex, weight should be used. Mode of action & pharmacological effects of both test & standard should be same so that their log DRC’S are parallel & their log-potency ratios can be easily compared.

5. TYPES OF BIOASSAYS 1. All/None Phenomenon /Quantal Bioassay/Direct end-point Bioassay. 2. Graded Response Bioassay. All/None Phenomenon /Quantal Bioassay/Direct end-point Bioassay: Response in the form of All/None[Either No response or maximum response produced] Example: Cardiac Arrest due to Digitalis in Guinea pig ileum. Hypoglycemic convulsions by Insulin in Mice. Head drop in Rabbits coz of paralysis of neck muscles by d-tb. LD50 Determination in mice / rats. Dose of the test & standard which gives All/None effect is estimated & then their potency ratios are calculated & compared.

6. Graded Response Bioassay It involves measurement & grading of responses of drug at different doses. Hence, method is known as “Graded Response Bioassay”. Example: Bioassay of Acetylcholine on Frog Rectus Abdominis muscle ,Where contraction of muscle at different doses is measured & graded.

7. Methods of Graded Response Bioassay: a] Matching Bioassay b] Bracketing Bioassay c] Interpolation Bioassay d] Multiple point Bioassay ( i ) Three-point Bioassay (ii) For-point Bioassay (iii) Six-point Bioassay Matching Bioassay In cases , where sample size is very small. Test response is matched against standard response if drug 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.

8. MATCHING BIOASSAY

9. BRACKETING BIOASSAY It involves bracketing of test response between one higher & one lower response of a standard drug. Interpolation of the bracket response on Dose-axis gives concentration of test sample. It’s a simple method . It requires very small amounts of sample.

10. INTERPOLATION BIOASSAY Log DRC plotted for standard Drug by Trial & Error method. Two / more responses of unknown(test) drug are plotted such that they fall in between the linear portion of log-DRC. Interpolation of such test responses @ dose axis gives X &X1 values . Antilog of X & X1 gives the concentration of test preparation.

11. Demerits : These graded interpolation bioassays are not reliable. The changes in tissue sensitivity overtime, dose timing, differences in mode of drugs application (T & S ) are not considered during assay. Statistical Evaluation is not possible.

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

13. Four-point assay: 2 standard responses ; 2 test responses are taken in to consideration. Log DRC of standard & test drug are plotted initially. 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. Test responses are selected such that they lie on linear portion of log DRC. They should be selected such that responses @ lower & higher doses of both standard & test preparations are similar. Ratios of test & standard doses.i.e ; t2/t1 & s2/s1 should be simple & same.

14. TH9X