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BIOASSAYS.pptx

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BIOASSAYS.pptx

  1. 1.  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  i.e.Observation of pharmacological effects on [1] living tissues, or cells [2] microorganisms or immune cells [3] animals  Hence micro bioassay and radioimmunoassay are also regarded as `bioassay'
  2. 2. Biological system can be  Intact organism  Isolated organs  Isolated tissues  Cellular systems e.g. RBC, WBC  Enzymes
  3. 3.  Active principle of drug is unknown  Active principle cannot be isolated, e.g. insulin, posterior pituitary extract etc  Chemical method is either  not available  if available, too complex or too costly  insensitive to low doses e.g. Histamine can be bioassayed in microgram conc.  Unknown Chemical composition, e.g. long acting thyroid stimulator  Sometimes the active substance to be assayed gets inactivated by interacting with chemicals in a chemical assay such as hormones  Chemical composition of drug variable but has same pharmacological action e.g. cardiac glycosides isolated from diff sources, catecholamines etc
  4. 4.  New Drug Development: Assessment of the pharmacological activity of NCEs (new chemical entities)  Measurement of concentration of known substances in biological fluids: bioavailability, drug-drug interactions  investigation of the function of endogenous mediators  Assessment of drug toxicity: Phenytoin, Digoxin, Theophylline are extremely potent with narrow therapeutic index  Diagnostic purposes: HCG in urine for pregnancy, AIDS virus  To measure clinical effectiveness of a treatment
  5. 5.  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  The standards are internationally accepted samples of drugs maintained and recommended by the Expert Committee of the Biological Standardization of W.H.O.  In India, standard drugs are maintained in Government institutions like Central Drug Research Institute, Lucknow, Central Drug Laboratory, Calcutta, etc.
  6. 6.  Active principle to be assayed should show the same measured response in all animal species  The degree of pharmacological response produced should be reproducible under identical conditions [e.g Adrenaline shows same rise in BP in the same species under identical conditions: wt, age, sex, strain / breed etc]  The reference standard must owe its activity to the principle for which the sample is being bioassayed  Activity assayed should be the activity of interest  Individual variations must be minimised / accounted for  Bioassay might measure a diff aspect of the same substance compared to chemical assay [Eg testosterone & metabolites
  7. 7. Potency The potency is a measure of drug activity expressed in terms of the amount required to produce an effect to given intensity. The more potent a drug the lower the amount required to produce the effect. In every type of bioassay, the relative potency of standard and unknown compounds are measured and threshold dose of each compound is calculated The term relative potency is used to express the biological activity of a sample preparation compared to a standard preparation.
  8. 8. Potency Potency Ratio The ratio of equipotent doses of the standard preparation and the unknown preparation under the conditions of the assay. Estimated Potency The potency calculated from assay data.
  9. 9. [1] Quantal Assays [Direct endpoint]  Elicits an ‘All or None’ response in different animals  e.g.  Digitalis induced cardiac arrest in guinea pigs  insulin-induced hypoglycemic convulsions in mice  Digitalis induced head drop in rabbits [2] Graded Response Assays [mostly on tissues]  Involves proportionate increase in the observed response following an increase in the dose  Unknown dose response measured on same tissue  e.g. contraction of smooth muscle preparation for assaying histamine
  10. 10.  Bracketing Method  Matching Bioassay  Interpolation Method  Multiple Point Bioassay
  11. 11.  The threshold dose producing a positive effect is measured on each animal and the comparison between the average results of two groups of animals (one receiving standard and other the test) is done.  e.g. bioassay of digitalis in cats  Potency is calculated as follows: Conc. of Unknown = Threshold dose of the Std X Conc. of Std. Threshold dose of the Test
  12. 12.  Simplest way of GRA, in which single response was taken by using any test drug concentration  response is bracketed between two responses (one higher and one lower) of the standard drug  Potency of test drug is directly calculated from concentration of standard drug or by extrapolation through dose response curve  Preferred when sample volume is too small  Limitation of the assay is large due to poor precision and reliability and also unable to calculate error
  13. 13.  Comparison of potency between unknown and standard drug: done by trial and error method  Response is matched at only one dose: no need of dose response curve  Requires very small sample volume  Disadvantages:  purely subjective  experimental error is not excluded out and  no sign of parallelism as it lacks dose response relationship  requires most sensitive tissue
  14. 14.  Concentration of unknown is extrapolated from the dose response curve graph  At first DRC of the standard drug is plotted then single or few responses of the test drug are plotted  The dose of the test drug which comes at the linear dose-response relationship is interpolated from the dose response plot
  15. 15.  Selection of 1 or more dose responses of test compound and these responses are compared with 2 or more responses of standards  The selection of the test doses must be in the linear portion of the dose-response plot of standard compound i.e. between 25% to 75%  Repeated response recording in graded response assays  minimize the tissue sensitivity error  improve the methodological errors  Further classified in to 3 point, 4 point, 6 point and 8 point assay
  16. 16.  Log dose response [LDR] curve plotted with varying conc of std drug solutions and given test solution  Select two std doses s1& s2 [ in 1:2 dose ratio] from linear part of LDR [S1 & S2]  Choose a test dose t with a response T between S1 & S2  Record 4 sets data [Latin square: Randomisation reduces error] as follows  s1 s2 t  t s1 s2  s2 t s1  s1 s2 t  Plot mean of S1, S2 and T against dose. Calculate  Relative potency [ M ] = [ (T –S1) / (S2-S1) ] X log (s2/s1)  Concentration of unknown compound = (s1/t) X antilog M
  17. 17. 3 point assay
  18. 18.  Log dose response [LDR] curve plotted with varying conc of std drug solution and given test solution  Select two std doses s1& s2 from linear part of LDR [Let the corresponding response be S1, S2]  Choose two test doses t1 & t2 with response T1 &T2 between S1 & S2  Record 4 data sets (16 responses) [Latin square: Randomisation reduces error]  s1 s2 t1 t2  s2 t1 t2 s1  t1 t2 s1 s2  t2 s1 s2 t1  Plot mean of S1, S2 and T1, T2 against dose.  Potency ratio [M] = [ (T1 –S1 + T2 –S2) / (S2-S1 + T2-T1) ] X log s2/s1)  Concentration of unknown compound = (s1/t1) X antilog M
  19. 19.  Generally not adopted for the experiment purpose because of the time consuming lengthy procedure  Responses obtained for the 6-point is ‘36’ and for 8-point is ‘64’ respectively  Advantage is reduced error and variability over other methods due to large number of responses and hence have greater specificity
  20. 20.  Less precise  Variable  Subjective  Time consuming  Technical skill required
  21. 21. Immunoassays are Sensitive Precise Rapid Convenient Small sample size required (100 µL)
  22. 22. Immune reaction = antibody (Ab)+ antigen (Ag)  Labelled with radioisotope: Radioimmunoassay (RIA)  Labelled with active enzyme: Enzyme immunoassay Drug specific Ab Ab-drug (bound drug) + Drug (free drug) =+ Ab-labeled drug + Labeled drug
  23. 23.  Also known as saturation analysis, developed in 1960 by Yalow and Berson for the assay of Insulin. Four requirements:  Antiserum  Labelled antigen (drug) with 125I  Standard unlabelled drug  Method to separate bound drug from free drug (Electrophoresis, gel filtration, adsorption techniques)
  24. 24.  Radioactive and non-radioactive drugs are indistinguishable chemicals  Drug and antibody react in the ratio of 1:1  AB is highly specific for drug and there are no cross reactions  125I in greater abundance  Better counting efficiency  Less penetrating so less hazardous  Longer T1/2 (60 days) Vs 131I (8 days)  Iodine is natural constituent of thyroid hormone  It can be introduced easily into peptide molecules Reasons for using 125I Vs 131I
  25. 25. Advantages  Highly Sensitive  Detects in Picomolar range Disadvantages  Expensive-Heavy Cost of Radioactive compounds  and Radioactivity counters  Can give false positive results with biological degraded  substances also.
  26. 26. Based on the principle of competitive displacement D-E D-E-Ab + Ab D D-Ab D: Drug, hormone or vitamin whose concentration to be determined E: Glucose-6-Phosphate Dehydrogenase, horseradish peroxidase
  27. 27. Advantages:  Less costly  No health hazards Widely used in qualitative and quantitative analysis of abused drugs in urine samples

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