1. Biological Evaluation Lecture - 4 Principles and Importance of BA’s Joseph O. Oweta B. Pharm (MUST)

2. Graded Dose Assays Graded assays are based on the observation that there is a proportionate increase in the observed response following an increase in the concentration or dose. When the response of a particular receptor-effector system is measured against increasing concentrations of a drug, the graph of the response versus the drug concentration or dose is called a graded dose- response curve The parameters employed in such bioassays are based on the nature of the effect the substance is expected to produce. For example: the study of blood pressure response in case of adrenaline.

3. Dose Response Curves Plotting the same data on a semilogarithmic concentration axis usually results in a sigmoid curve, which simplifies the mathematical manipulation of the dose-response data. The efficacy (E max ) and potency (EC 50 or ED 50 ) parameters are derived from these data. The smaller the EC 50 (or ED 50 ), the greater the potency of the drug.

4. Dose Response Curves

5. Efficacy Efficacy—often called maximal efficacy—is the greatest effect (E max ) an agonist can produce if the dose is taken to very high levels. Efficacy is determined mainly by the nature of the drug and the receptor and its associated effector system. It can be measured with a graded dose-response curve but not with a quantal dose-response curve. By definition, partial agonists have lower maximal efficacy than full agonists

6. Potency Potency denotes the amount of drug needed to produce a given effect. In graded dose-response measurements, the effect usually chosen is 50% of the maximal effect and the dose causing this effect is called the EC50 (A and B). Potency is determined mainly by the affinity of the receptor for the drug and the number of receptors available. In quantal dose-response measurements, ED50, TD 50, and LD50 are also potency variables (median effective, toxic, and lethal doses, respectively, in 50% of the population studied). Thus, potency can be determined from either graded or quantal dose- response curves (next slide), but the numbers obtained are not identical.next slide

7. Potency

8. Matching Bioassay It is the simplest type of the bioassay. In this type of bioassay, response of the test substance taken first and the observed response is matched with the standard response. Several responses of the standard drug are recorded till a close matching point to that of the test substance is observed. A corresponding concentration is thus calculated. This assay is applied when the sample size is too small. Since the assay does not involve the recording of concentration response curve, the sensitivity of the preparation is not taken into consideration. Therefore, precision and reliability is not very good.

9. Matching Bioassay Firstly responses of the test of a particular dose is taken It is matched with the dose of the standard (whose strength is known) by trial & error method Done till a closed matching is observed. Corresponding concentration calculated. Potency ratio of the two can be approximately found & strength of the unknown test solution can be calculated Eg. histamine bioassay, posterior pituitary assay on the rat uterus

10. Bracketing Bioassay Used when test sample is too small Response of test is bracketed b/w two responses (greater & smaller) of standard substance. Strength of unknown found by simple interpolation of this bracketed response on the dose axis Precision & reliability is poor.

11. Bracketing Bioassay

12. Matching/Bracketing Advantages: Faster Can be completed when amount of test drug available is small Does not involve complicated calculations Disadvantages: Match is subjective Exact match may not always be possible No evidence of parallelism/ discrimination Does not permit calculation of variation.

13. Interpolation bioassay: Bioassays are conducted by determining the amount of preparation of unknown potency required to produce a definite effect on suitable test animals or organs or tissue under standard conditions. This effect is compared with that of a standard. 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.

14. Assignment Read and Make notes about Multiple point assays: – Three point bioassay [2+1 dose assay] – Six point bioassay (3 + 3 assay)

15. Errors in Bioassays 1.Biological Variation – errors in response due to some fault in the test subject 2.Methodological Error – due to faulty method selection or incorrect procedure

16. Biological Variation ReasonCorrection Down regulation of receptorsAvoid pre exposure of test subjects Loss of sensitivityChange test subject Variation in Lab ConditionsShould be maintained constant

17. Methodological Error ReasonCorrection Lack of standardisation of procedureStandardize procedure before hand Set up of apparatusValidation of instruments before use Tissue isolation and preparation for experiment Minimise handling Drug preparation and dilutionMix slowly while diluting avoid vigorous shaking

18. Any Questions Next Lecture: We shall discuss Animal ethics whilst considering Institutional ethics committee, Euthanasia, Animal handling, disposal of animals after experiment and post experimental care