GATE : Bioassay
From Pharmpedia
BIOASSAY
In several biological experiments, there is a strong correlation between the dose of a drug and the response elicited. If dose is increased systematically in an isolated tissue or single animal, a graded response may be obtained. At first there will be a range of doses so low that no response is seen. Then a higher range of doses elicits responses of increasing magnitude, and finally a maximal response may be attained which cannot be exceeded at any dose. If log dose is plotted on the X axis and response on the Y axis, a symmetrical sigmoid curve is characteristically obtained. The central portion of this curve is linear. This means that, over a considerable range of doses, increasing the dose by constant multiples causes equal linear increments of response.
The log dose-response curve may be regarded as a cumulative normal frequency distribution.
When half of the population responds to produce 50% of the maximal response, we call this median effective dose, the ED50.
The slope of the log dose-response curve reflects the variance of sensitivities of the responsive units, the steeper the curve,
the more homogeneous are the responsive units, i.e, the smaller is their variance.
Since the interval µ±σ⌐/2 includes 38% of the area of the normal curve (19% on each side of µ), σ⌐ can be estimated directly as the distance on the log-dose axis between the ED31 and the ED69.
Often the responsive units are whole animals, and the measured responses are quantal, in that an animal either responds or does
not. Thus, in toxicity tests groups of animals are exposed to different drug doses and the percent showing the toxic effect at
each dose is noted. In clinical trials the measure of response at each dose level may be the percent of patients cured. If the
response measured is death, the curve is known as a log dose-mortality curve, and the median lethal dose is called the LD50.
The two features which characterize any log dose-response curve are its position on the log-dose axis (given by the ED50) and its slope at the ED50. If two drugs act by the same mechanism upon the same responsive units, but differ only in potency, the slopes of their curves must be the same.
Different slopes imply different mechanisms of action.
If the two curves are equidistant from each other in the horizontal direction at all response levels, it is meaningful to state
the difference in potency without further qualification. If the two curves are not parallel, a potency comparison is meaningful
only if the particular response level (eg. The ED50) is specified.
- Bioassay is defined as the estimation of the concentration or potency of a substance by measurement of the biological response that it produces.
- Uses of Bioassay :
- To measure the pharmacological activity of new or chemically undefined substances.
- To investigate the function of endogenous mediators
- To measure drug toxicity and unwanted effects.
- Pharmaco economics and pharmaco epidemiology are subjects which have to do with the study of socio-economic groups. When the effect of the use of a drug on the health care costs, social costs and disability costs is studied it is pharmaco economics. When the effect of the use of a drug on the disease prevalence in a socio-economic group is studied, it is pharmaco epidemiology.
- Biological assays are designed to measure the relative potency of two preparations, usually a standard and an unknown.
- The best kind of standard is the pure substance.
- If S stands for standard, U for unknown.
- A bioassay must provide an estimate of the dose or concentration of U that will produce the same biological effect as that of a known dose or concentration S.
- If the log dose vs effect curves of Sand U are parallel, the ratio M of equiactive doses will not depend on the magnitude of response chosen. So M is an estimate of the potency ratio of the two preparations.
- A comparison of the magnitude of the effects produced by equal doses of S and U does not provide as estimate of M.
- The design of bioassays is aimed at
- Minimizing variation
- Avoiding systematic errors resulting from variation
- Estimation of the limits of error of the assay result.
- In a parallel line assay.
- X axis has logarithmic dose scale
- The curves of standard and unknown are normally parallel.
- The potency ratio M is is determined by the horizontal distance between design is a
- The minimal design is a 2+2 assay (two doses of standard and two doses of U are used)
- In practice, most bioassays will give results whose 5% confidence limits lie within 20% of the true value.
- If the lines are not parallel, it means that the two drugs are having two different mechanisms of action. In this case, it is not possible to define the relative potencies of S and U, clearly, in terms of a simple ratio.
- Graded response means, a particular effect seen at different levels. Ex:change in blood glucose concentration, contraction of a strip of smooth muscle, change in the time taken for a rat to run a maze.
- All or nothing response : There is no gradation here; either the response or effect is there, or it is not there. Ex: death of animal, loss of righting reflex, success in maze – running within a stipulated time. This is also known as quantal response.
- The shape and slope of such a curve is governed by the individual variation between animals – the more uniform the population, the steeper the curve, and the more precise the assay.
- With graded responses, the steepness of the dose-response curve is a property of the test system and has nothing to do with biological variation.
- Clinical trials aim to measure therapeutic effectiveness and constitute an important and highly specialized form of biological assay.
- In a bioassay involving four human volunteers, the results showed morphine to be 13 times as potent as codeine.
- Responses to drugs tend to vary proportionally to log dose rather than to dose, so dose-response correlations are routinely plotted with log-dose rather than dose on the X axis.
- The details of bioassay methods to be followed are given in the Pharmacopoeias (IP, BP etc.).For many drugs there are more than one bioassay methods. The assay parameters recorded for some drugs are given below.
Adrenaline. Blood Pressure raising effect in the cat or dog. Noradrenaline. Rise of blood pressure in the spinal cat. Histamine. Contraction of the isolated guinea pig ileum. Insulin. Hypoglycaemic convulsions in mice. Acetylcholine. Contraction of the frog’s rectus adbominis muscle. d-Tubocurarine. Rabbit head drop due to paralysis of neck muscles. Androgen. Growth of the capon’s comb. Oestogen. Cornification of the vagina in rats.
Digitalis Death due to cardiac arrest in systole in the guinea pig. There are four methods of Bioassay : Method 1 : Threshold dose measured on each animal (Direct end-point assay). Method 2 : Responses Recorded or measured (Graded response assay). Method 3 : Percentage of positive effects measured (Quantal assay or All or None essay). Method 4 : Microbiological assays : These methods are used for assaying antibiotics. The test preparation is compared with the standard preparation for its inhibitory effect on particular organisms, sensitive to the agent.
Author
This article is contributed by Smt. Prof. Jayanti Vijaya Ratna .
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