Introduction
What is bioassay?
Defined as the estimation of the concentration or potency of a substance (drugs, hormones, vitamins, toxins, and antitoxin) by measurement of the biological response that it produces (by means of biological indicators , in a live animals, isolated tissues or cell line).
Biological indicators
Examples
*Blood pressure *Blood glucose *Muscle contraction *Inhibition of growth of microorganisms
Screening of drugs
*It means thorough investigation of substance for pharmacological activity and evaluation of this activity i.e. scanning and evaluation. The main purposes of screening are to determine whether the new substance are worthy for further attention and to indicate which among them have the most interesting pharmacological properties.
Types of screening
Blind screening
Simple screening
Programmed screening
Types of screening
A- Simple screening
It involves the use of one or two simple tests to find substances having a particular property. For example, a single test for conc. of glucose in blood can be used to screen compound for hypoglycemic activity.
Types of screening
B- Blind screening
*It is used to detect to the pharmacological activities of new drugs whose pharmacological activity is unknown. *The chief purposes is to demonstrate whether these new drugs are worthy of further attention
Types of screening
C- Programmed screening
*It is used when a new drug of specific type is to be screened for some pharmacological effects. Examples are screening of certain drugs on the cardiovascular system, CNS, kidney, blood etc. *It includes the use of quantitative assay of the compounds and their comparison with standard drugs that are quite active representative members of their pharmacological class. *It also provides indications of potential side effects
whether the new drug is ganglionic blocker, adrenergic neuron blocker or alpha receptors blocker
This is usually carried out to illustrate the type of receptors the drug act on.
*Sex
Difference in drug response among different sex is attributed to difference in the level of metabolizing enzymes
*Age
The level of metabolizing enzymes varies with age and some enzymes may be lacked in newly born animal.
*Diseases
The hypersensitivity to catecholamine action on the CVS is increased in hyperthyroidism.
*Environmental
Such as temperature, seasons, nutrition, light and isolation of animal. For example, the convulsive action of insulin in mice is greatly influenced by temperature
Principle of Bioassay
*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.
Principle of Bioassay
*Comparisons are best made on the basis of doseresponse curves, *Different approaches can be applied according to the level of biological organisation at which the drug effect needs to be measured. Approaches range through molecular and chemical techniques, in vitro and in vivo animal studies, and clinical studies on volunteers and patients
Bioassayed by
(1) Matching or bracketing method or (2) Graphical method
The drug is slowly infused into the animal and the moment the heart stops beating and blood pressure falls to zero, the volume of fluid infused is noted down.
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. (The height of contraction is measured and plotted against the log-dose). *The concentration of test sample is determined by the same formula as mentioned before.
Graphical method
The characteristic of logdose 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
Graphical method
*Advantage of this method is that, it is a simple method and chances of errors are less if the sensitivity of the preparation is not changed.
Bioassay of Antagonists
*Commonly used method for the bioassay of antagonist is simple graphical method. *The responses are determined in the form of the percentage 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.
Bioassay of Antagonists
*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 ten min till recovery is obtained. *The higher dose of standard antagonist is added and responses are taken as before.
Bioassay of Antagonists
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. The percentage inhibition is calculated, plotted against log dose of antagonist and the concentration of unknown is determined as usual.
*Selectivity
It is ability of the test drug to selectively use a certain signal transduction pathway to act on the same receptor (e.g., histamine selectively acts on histaminic receptors and not muscarinic receptors).
*Sensitivity
It is the ability of the animal or tissue used in the bioassay to respond to small amounts of the test drug (e.g., in the assay of histamine, the guinea pig ileum is used rather than the rabbit intestine due to the presence of high amounts of histaminase enzyme in the rabbit intestine that catalyze the inactivation histamine).
*Accuracy
It is the degree of closeness of measurements of a quantity to its true value.
*Precision
It is the degree to which repeated measurements under unchanged conditions show the same results.
Dose-response curve
A dose-response curve is a simple X-Y graph relating
The concentration of the agonist (X-axis) The agonist response (Y-axis)
The response may be physiological or biochemical such as enzyme activity, secretion of a hormone, heart rate or contraction of a muscle.
Dose-response curve
The dose-response curve is obtained by gradual increasing the dose of the drug until reaching two successive doses giving the same response (maximal response). The range of increasing effect with increasing dose is termed the "submaximal" range
Dose-response curve
Dose-response curve
The Emax is the maximum possible effect for the agonist.
The concentration of the drug at which the % of the Emax is termed half maximal effective concentration and is abbreviated as EC50.
Dose-response curve
In most cases, a log scale is used for the dose axis rather than a linear scale; the reason is to include a wide range of doses, as shown in the semi-log paper
4-point assay of acetylcholine using the isolated rabbit intestine In the 4-point assay, 2 doses of standard acetylcholine solution (S1 and S2) are randomized with 2 doses of test acetylcholine solution (T1 and T2) according to the following 3 criteria: 1. All doses of both the standard and the test should be submaximal (i.e. the response is directly proportional to the dose).
4-point assay of acetylcholine using the isolated rabbit intestine 2. The volume of T1 is selected to give a response approximately equal to that of S1. 3. The volume of T2 is mathematically calculated to fulfill the following ratio:
4-point assay of acetylcholine using the isolated rabbit intestine N.B. The volume of the selected doses should be within 0.1 to 1 ml because a volume less than 0.1 ml is not accurately measured and more than 1 ml will change the balance of the physiological solution in the organ bath.
4-point assay of acetylcholine using the isolated rabbit intestine 5. The four selected doses (S1, S2, T1& T2) are then randomized using the Latin square design. 6. Measure the responses produced after randomization and calculate the mean of these responses for each dose in mm.
7. Calculate the concentration of test acetylcholine solution using both, graphical and mathematical methods.
Graphical method
Plot the results using a semi-log paper, where the X-axis has a log scale that runs from 1 to 10 (as previously shown) and the Y-axis has a normal scale.
4-point assay of acetylcholine using the isolated rabbit intestine N.B. The line representing the standard is parallel to the line representing the test. This is because both the standard and the test have the same mechanism of action (they are the same drug but in different concentrations).
Applications of the affinity constant KB 3- To identify a receptor in a tissue: e.g.: if the KB of atropine in a tissue was found to be = 1x109, therefore this tissue contains muscarinic receptors.