Bioassay & Pharmacological screening

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

Applications of bioassay in pharmacology

*Determination of drugs potency. *Screening of new agents isolated from plants, animals or chemical labs and find their field of activities. *Determine the therapeutic advantage of one drug over anther treatments. *Determination of the pharmacological activities of a new drug. *Establishment of SAR

Comparison between Bioassay and Chemical assay

*Bioassay is less precise, more time consuming and more expensive than chemical assay. *In bioassay, active constituent and its structure do not have to be known. *Bioassay is more sensitive than chemical assay (detect even small amount of the drug).

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

Steps for biological screening

Neuropharmacological tests
To detect
Sedative, hypnotic, tranquillizer, psychomotor stimulant, muscle relaxant, analgesic and antipyretic

Steps for biological screening

Neuropharmacological tests
Mice are usually used here because they are small, easy to handle, economic, mammals and have long life span.

Steps for biological screening

Neuropharmacological tests
Signs to be observed
Consciousness, awareness, motor activity, pain response and excitability. Other responses should be evaluated like pupil size, secretions, heart rate, respiration, and writhing

Steps for biological screening

Cardiovascular tests
To study the action of new drug on blood pressure and heart rate usually on anesthetized Rat OR Cat

Steps for biological screening

The cat nictitating membrane *It is an additional eye lid that composed
of smooth muscle innervated by post-ganglionic sympathetic nerve fibers. It has alpha and beta subtype of receptors.

*Nictitating membrane is attached to a lever

for recoding its contraction.

*By the aid of this test we can determine

whether the new drug is ganglionic blocker, adrenergic neuron blocker or alpha receptors blocker

Steps for biological screening

Tests on isolated organs
EX
Guinea pig ileum, intestine Rat uterus, aorta

This is usually carried out to illustrate the type of receptors the drug act on.

Factors that can affect the pharmacological response to drugs

*Species and strain of animals

Differ in biochemical, functional and morphological features thus their response to the drug will vary quantitatively or qualitatively.
Example, morphine
CNS depressant in rabbit and human CNS stimulant in cats and dogs

Factors that can affect the pharmacological response to drugs

*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.

Factors that can affect the pharmacological response to drugs

*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

Methods of Bioassay for Agonists

An agonist may produce Graded response or Quantal response
The response is proportional to the dose and response may lie between no response and the maximum response. The response is in the form of "all or none", i.e. either no response or maximum response

Bioassayed by
(1) Matching or bracketing method or (2) Graphical method

End point method

End Point Method

Here 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

End Point Method

Ex: Bioassay of digitalis in cats

The cat is anaesthetized with chloralose

Blood pressure is recorded

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.

End Point Method

Ex: Bioassay of digitalis in cats
Two series of such experiments-one using standard digitalis and the other using test preparation of digitalis is done and then potency is calculated as follows:

Matching point or Bracketing method

Here a constant dose of the standard is bracketed by varying dose of sample until an exact matching between the standard dose responses and the particular dose response of the sample is achieved.

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.

Other methods which are based on the dose-response relationship include

3 point assay method 2 doses of the standard and one dose of the test 4 point assay method 2 doses of standard and 2 doses of the test 6 point assay method 3 doses of standard and 3 doses of the test

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.

4-point assay of acetylcholine using the isolated rabbit intestine

Criteria of a good biological assay

*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).

Criteria of a good biological assay

*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).

Criteria of a good biological assay

*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

Principle of the assay:

Acetylcholine acts on muscarinic M3 receptors in the isolated rabbit intestine causing contractions. The magnitude of these contractions is proportional to the concentration of acetylcholine in the organ bath (graded response). Therefore, the concentration of a test solution of Ach can be determined by the bioassay of this test solution against a standard solution of Ach of known concentration. For this purpose, the 4- point (2+2) bioassay is used.

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

After selecting S1, S2, T1 & T2, randomization of these doses is performed according to the Latin square design in which each dose is added 4 times according to the following sequence:

4-point assay of acetylcholine using the isolated rabbit intestine

Practical steps:
1- Set a piece of rabbit intestine in the organ bath filled with physiological solution (Tyrode). Adjust the temperature to 37 oC and adjust the aeration as a fine steady stream of air. 2- Select 2 sub-maximal doses of standard acetylcholine solution (S1 & S 2 by testing the response of several ascending doses of the standard.

4-point assay of acetylcholine using the isolated rabbit intestine

S1 & S2 should fulfill the following criteria: They should be submaximal. They should produce reasonable responses. The difference in the response between them should be significant (approximately 1 cm or more).

4-point assay of acetylcholine using the isolated rabbit intestine

3. Repeat adding the 2 selected standard doses (S1 & S2) to check for the reproducibility of the response. 4. Select 2 doses for test acetylcholine (T1 & T2) so that T1 gives a response as S1, then T2 is calculated mathematically as mentioned before.

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.

4-point assay of acetylcholine using the isolated rabbit intestine

7. Calculate the concentration of test acetylcholine solution using both, graphical and mathematical methods.

4-point assay of acetylcholine using the isolated rabbit intestine

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).

4-point assay of acetylcholine using the isolated rabbit intestine

4-point assay of acetylcholine using the isolated rabbit intestine

Relative potency estimation

The doses of standard and sample that yield some selected magnitude of response in a bioassay can be defined as equally effective dose Thus, at any given magnitude of response, the potency of a sample relative to a standard is the ratio of the doses of standard and sample needed to elicit that response Relative potency = dose std/dose sample

4-point assay of acetylcholine using the isolated rabbit intestine

Relative potencies have traditionally been calculated as the ratio of the EC50s of the two compounds being compared:

Relative potency = EC50A/EC50B

Bioassay of atropine using the affinity constant method

Atropine is a reversible competitive antagonist to acetylcholine at the muscarinic receptors.

This competition depends on 2 factors:

1. concentration of atropine at the receptors 2. affinity of atropine to the receptors.

Applications of the affinity constant KB

1- To determine the potency of the antagonist in terms of "pA2 value", which is the negative logarithm of the molar concentration of the antagonist required to double the dose of the agonist to produce the same response as in the absence of the antagonist. N.B. the higher the pA2 value the higher the potency of the antagonist. pA2 = Log KB For atropine, the KB at muscarinic receptors = 1x109 . Therefore, the pA2 for atropine = log 1x109 = 9

Applications of the affinity constant KB

2-To determine the site of action of an unknown antagonist:
e.g.: an unknown antagonist was tested on two preparations: Rabbit intestine using acetylcholine (KB atropine at muscarinic receptors = 1x109 and KB unknown at muscarinic receptors = 1x103; pA2 atropine = 9, pA2 unknown = 3). Guinea pig ileum using histamine (KB mepyramine at histaminic receptors = 2x109 and KB unknown at histaminic receptors =3x109; pA2 mepyramine = 9.30, pA2 unknown = 9.48). Therefore, the unknown antagonist blocks histaminic receptors with very weak antimuscarinic action.

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.

Applications of the affinity constant KB

4- To determine the concentration of an antagonist: e.g.: during the bioassay of atropine using the isolated rabbit intestine, the following results were obtained: a dose of 0.6 ml of Ach produced a response in presence of 0.5 ml atropine, similar to that produced by 0.2 ml of Ach in absence of atropine. Knowing that the molecular weight of atropine sulphate is 700, the capacity of the organ bath is 100 ml, and the affinity constant KB of atropine at muscarinic receptors is 1x109, calculate the concentration of atropine in the provided sample in g/ml.