mbe part 1 (1)

7/31/2019 MBE PART 1 (1)

1/44

20302: Pharmacometrics and Methods of Biological Evaluation of Drugs

Date:

PRACTICAL-1

Introduction to Bioassay

AIM: Introduction to bioassay of drugs.

DEFINITION:

It is an estimation of the potency of an active principle in a unit quantity of preparation and

measurement of the concentration of the substance in a preparation using biological

method (i.e. observation of pharmacological effect on living tissues, microorganisms or

immune cells or animal) is known as biological assay or bioassay.

IMPORTANCE OF BIOASSAYS:

Bioassays are essential in the development of new drugs. In the preclinical assessment of a

new compound, the biological activity is compared with that of known compounds using

appropriate test systems. The precision, reliability and reproducibility of bioassay depend

on the proper selection of the tissue or method with highest selectivity and sensitivity for

the drug. In spite of the tremendous advancement in the analytical chemistry and modern

instrumentations, bioassay procedures continue to be used as successful tools not only in

the estimation of bioactive substances but also for the discovery of biologically active

substances.

Bioassays are generally employed:

When active principle of drug is unknown or cannot be isolated.

When a chemical assay for the substance is not available or interacting with chemicals

as the case with hormones inactivates the substance. Chemical method is too complex,

insensitive or requires higher dose.

When the quantity of the sample is too small. In such situation a matching type of

bioassay is conveniently done to compare the biological response with the standard drug.

To estimate the concentrations of active principles present in the tissue extracts, the

endogenous mediators like acetyl choline, 5-HT, prostaglandins

To measure the pharmacological activity of new or chemically unidentified substances

To measure drug toxicity and

When bioassay is more sensitive than chemical assay.

The purpose of bioassay is to ascertain the potency of a drug and hence serves as the

quantitative part of any screening procedure. Other purpose of bioassay is to standardize

the preparation so that each contains the uniform specified pharmacological activity, serve

as pointer for the commercial production of drugs, and help diagnosis of various

conditions.

PRINCIPLE OF BIOASSAY:

The basic principle of bioassay is to compare the substance with the international

preparation of the same and to find out how much test substance is required to produce thesame biological effect, as produced by standard. The problem of biological variation must

M. Pharm II-semester (Pharmacology) - 1 -

ANAND PHARMACY COLLEGE

7/31/2019 MBE PART 1 (1)

2/44


be minimized as far as possible. For that one should keep uniform experimental conditions

and assure the reproducibility of the responses.

USE OF STANDARDS:

Bioassays are designed to measure relative potency of two preparations, usually a standard

and an unknown. It is unsatisfactory to designate a unit of particular drug at that amountand causes a particular effect because biological effects vary from animal to animal, time

to time & from lab to lab. Use of standard substance for comparison also helps in solving

problems arising from biological variations. The observed response/effect of the unknown

would be always relative to the effect that produced by a standard substance. The standard

substance is a pure substance and in official bioassays it refers to pharmacopoeial

standards. In case of hormones, biological products and vaccines it is often necessary to

establish the standard response of the standard substances against which unknown samples

can be calibrated.

DISADVANTAGES OF BIOASSAY:

Less accurate

Less elaborate

More laborious

More troublesome

More expensive

PRECAUTIONS IN BIOASSAYS TO MINIMIZE BIOLOGICAL VARIATIONS:

All the experimental conditions should be constant.

The response studied should be reproducible.

The biological response being studied should be sensitive to the drug.

The animals should be of same species, strain, approximate of same age and weight

and sex. Also should be kept on a similar diet and housed under similar conditions.

METHODS OF BIOASSAY FOR AGONISTS:

An agonist may produce two types of response.

[1] Quantal Response:

Quantal means that the response is in the form of all or none i.e. either no response or

maximum response. The drugs producing quantal effect can be bioassayed by 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 is done. For ex. Bioassay

of digitalis in cats. Here the cat is anaesthetized with chloralose and its blood pressure is

recorded. The drug is taken slowly infused into the animal and the moment the heart stopsbeating and blood pressure falls to zero, the volume of fluid infused is noted down. Two



7/31/2019 MBE PART 1 (1)

3/44


series of such experiments-one using standard digitalis and other using test preparation of

digitalis is done and then potency is calculated as follows:

Conc. of Unknown = (Threshold dose of Std. /Threshold dose of test) X Conc. of Std.

In case, if it is not possible to measure individual effective dose or if animals are not

available, fixed doses are injected into groups of animals and the percentage of mortality ateach dose level is determined. The percentage of mortality is taken as the response and

then the comparison is done in the same way as done for graded response.

[2] Graded Response:

Graded response means that the response is proportional to the dose and response may lie

between no response and the maximum response. Graded response assays are based on the

proportionate increase in the response in the response observed with an increase in the

concentration or the dose of the drug. The parameter employed in such bioassays are based

on the nature of the effect, the drug or substance is expected to produce. For ex.Contraction of smooth muscle of rat ileum for bioassay of acetylcholine, Relaxation of

smooth muscle of rabbit ileum for bioassay of Adrenaline. The drugs producing graded

responses can be bioassayed by:

(A) Graphical method or interpolation method

(B) Matching or bracketing method

(C) Multiple point method

The choice of the procedure or method depends upon precision or accuracy of assay, the

quantity of test sample available, the availability of experimental animals.

(A) 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. In simpler design, 5-6

responses of the graded doses of the standard are taken and then two equiactive responses

of the test sample are taken. The height of concentration is measured and plotted against

the log-dose. The dose of standard producing the same response as produced by the test is

read directly from the graph and the concentration of test sample is determined by the

following formula.

Conc. of Unknown = (Threshold dose of Std. /Threshold dose of test) X Conc. of Std.

The characteristic of log-dose 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.

Advantages:

It is a simple method.

Chances of errors are less if the sensitivity of the preparation is not changed.



7/31/2019 MBE PART 1 (1)

4/44


(B)Matching Method:

In this method a constant dose of the test is bracketed by varying doses of standard till the

exact match is obtained between test dose and the standard dose. Initially, two responses of

the standard are taken. The does are adjusted such that one is giving response of

approximately 20% and other 70% of the maximum. The response of unknown that lies

between two responses of standard dose is taken. The panel is repeated by increasing ordecreasing the doses of standard till all three equal responses are obtained. The dose of test

sample is kept constant.

In the end, a response of the double dose of the standard and test that match each other are

taken. These should give equal responses. Concentration of the test sample can be

determined as follows:

Conc. of Unknown = (Dose of Std. / Dose of Test) X Conc. of Std.

Advantage:

Useful when sensitivity is not stable.

Limitations:

It occupies a larger area of the drum as far as tracings are concerned.

The match is purely subjective, so chances of error are there and one cannot determine

them.

It does not give any idea of dose-response relationship.

Method is not accurate and not reliable.Ex. Bioassay of histamine on guinea pig ileum is preferably carried out by this method.

(C) Multiple point Bioassay:

These methods include 3 point, 4 point, 5 point and 6-point methods. In these methods, the

responses are repeated several times and the mean of each is taken. Thus, chances of error

are minimized in these methods.

In 3-point assay method, 2 doses of the standard and one dose of the test are used. Initially

a graded dose response curve for the standard drug is taken. From this response two doses

of the standard drug S1 & S2 are selected. The two doses should preferably be in the ratio

of 1:2.

The test dose is fixed in such a way that it gives the response between the responses

produced by S1 & S2. These three selected doses are repeated by the Latin Square design

method i.e. S1, S2, T S2, T, S1 T, S1, S2.

In order to avoid bias. The mean responses are calculated and plotted against log-dose and

amount of standard producing the same response as produced by the test is determined

mathematically:



7/31/2019 MBE PART 1 (1)

5/44


n1 T S1 n2

Conc. of Unknown= Cs X ---- X antilog ------------ X log ---- X Dilution Factor

t S2 S1 n1

Where, n1 = Lower std. dose

n2 = Higher std. dose

t = Test dose

S1 = Response of n1

S2 = Response of n2

T = Response of t

Cs = Conc. of Std.

In 4 point method two doses of standard and two doses of test, in 5 point method three

doses of standard and two doses of test, in 6 point method three doses of standard and three

doses of test are used. Similarly one can design 8-point method also.

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 effectas produced by the test.

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 response of agonist is repeated every ten min. till recovery is obtained. The higher

dose of standard, antagonist is added and responses are taken as before, three to four doses

of the standard. Antagonist is used and than one to two doses of test sample of the

antagonist is used similarly. The percentage inhibition is calculated, plotted against log-

dose and the concentration of unknown is determined as usual.

BIOASSAY ON SOME IMPORTANT DRUGS:

Depending upon pharmacological action of various drugs, different, preparations may be

used. Following chart gives different preparations and the pharmacological activity for

which a particular drug is assayed:

Sr.

No.DRUGS PREPARATION ACTIVITY ASSAYED



7/31/2019 MBE PART 1 (1)

6/44


01 Digitalis Cat blood pressure

Guinea pig Blood pressure

Fall in blood pressure or

stoppage of heart and death

02 Adrenaline Blood pressure of the spinal cat Rise in blood pressure

Isolated rabbit duodenum, Isolated

Caucus of fowl, Isolated rat uterus

Inhibition of tone

03 Nor adrenaline Blood pressure of the pitched cat. Rise of B. P04 Acetylcholine Isolated rectus abdominus of frog,

Rat ileum and leech dorsal muscle

Isolated mouse heart Inhibition of cardiac

contractions.

Rat / Cat blood pressure Fall in blood pressure.

05 Histamine Isolated, atropinized terminal ileum

of guinea pig.

Contractile effect.

Anaesthetized and atropinized cat. Fall in blood pressure.

06 5 Hydroxy-

Tryptamine

Isolated atropinized rat uterus,

Isolated Terminal colon of rat,Isolated fundus Strip of rat stomach,

Isolated heart of cat

Contractile effect

Perfused rabbit ear Constriction of blood vessels

07 Curariform

drugs

e.g. d-tubo-

curarine

Rabbit Dropping of head

Rat diaphragm with phrenic nerve,

Cat Gastrocnemius muscle with

sciatic nerve

Inhibition of the contractile

effect

08 Heparin Sulfated whole blood of ox with

thrombokinase extract and acetonedried ox brain.

Prolongation of blood clotting

time.

09 Antibiotics Suitable micro-organism grown on

suitable nutrient agar medium

Inhibition of growth of

microorganism.

10 Vitamin D. Rats maintained on richetogenic diet. Alleviation of rachitic stage

11 Insulin Rabbits Lowering of blood-sugar level

Mice Convulsions and/or death due

to hypoglycemia

Isolated rat diaphragm Increase in glycogen content.

Rats epididymal fat Increased metabolism of

glucose, indicated by increase

CO2 production.

12 Oxytocin Adult cockerel. Vasodepressor activity.

Isolated rat uterus. Contractile effect

Rabbits (female) Ejection of milk from

mammary duct

13 Vasopressin Rat blood-pressure Vasopressor activity.

14 Growth

hormone

Hypophysectomized rats. Gain in weight, Increase in

width of epiphyseal cartilage

15 Gonadotrophin(FSH)

Hypophysectomized male rats Increase in testicular weight.Hypophysectomized Female rats Increase in weight of ovaries.

16 Gonadotrophin Immature male rats. Enlargement of prostate glandM. Pharm II-semester (Pharmacology) - 6 -


7/31/2019 MBE PART 1 (1)

7/44


(LH)

17 Gonadotrophin

(FSH & LH)

Immature female rate. Increase in weight of uterus.

18 *Prolactin Cloves of pigeons. Increase in weight of crop sac.

Female guinea-pig or rabbit Secretory changes in

mammary gland.Female rats. Lengthening of estrous cycle

and function of corpus

luteum.

Hypophysectomized rat. Inhibition of estrogen upon

vaginal smear

19 *Corticotrophin Hypophosectomized rats. Depletion of ascorbic acid

from adrenal gland

20 *Thyrotropin Mice or rats. Release of previously

administered 131I (Iodine)

from thyroid gland.

21 *Androgen Castrated capon Increase in size of comb

Castrated male rat. Increase in weight of prostate

gland and seminal vesicles.

Castrated male rats. Increase in weight of levator-

ani muscles.

22 Estrogen Rat or mouse (Female) Increase in weight of uterus.

23 Progesterone Sexual immature rabbits Proliferative changes in

endometrium of uterus or

Increase In Carbonicanhydrase-activity in uterus.

*Radioimmunoassay or radio receptor assay methods are also available

CURRENT STATUS OF BIOASSAY:

Above-mentioned discussion is an overview of bioassay, which is prevailing, in various

academic institutions. However, with advent of technology, availability of advanced

sophisticated and more reliable analytical method the scenario for bioassay has changed

dramatically. If one reviews the emphasis of bioassay in pharmacopoeia published before

1980 as compared to those published recently. It will be clear that:

There are very few drugs which are now recommended to be assayed by biological

method.

Most of drugs, which were assayed by biological methods, are now being

recommended to be assayed by chemical methods.

Newer drugs have been included for which bioassay recommended.



7/31/2019 MBE PART 1 (1)

8/44




Teachers sign

7/31/2019 MBE PART 1 (1)

9/44


Date:

PRACTICAL-2

Bioassay of agonist-acetylcholine by Graphical Method using rat ileum preparation

AIM: To find out the concentration of unknown sample of acetylcholine by graphical

method using rat ileum.

PRINCIPLE:

This method is based on the assumption of the dose-response relationship. Acetylcholine

produces a dose dependent contraction of rat ileum smooth muscle. Graded responses of

acetylcholine are taken and two equipotent responses to unknown sample are taken. In

simpler design, 5-6 responses of the graded doses of the standard are taken and then two

equi-active response of the test sample are taken. The height of the contraction is measured

and Log-dose-response curve is plotted. The dose of standard producing the same response

as produced by the test sample is directly read from the graph and the concentration ofunknown is determined by the formula.

ADVANTAGES:

Most simple method.

Chances of error are less if the sensitivity of the preparation is not changed.

REQUIREMENTS:

Animals : Rat(of either sex weighing between 200-250g.)

Drugs : Acetylcholine (10 g/ml, 100 g/ml, 1 mg/ml)

Apparatus : Students physiograph, Mammalian isolated organ bath, organ tube,

Thermostat, isotonic frontal writing lever, recording drum, aeration

tube cum Tissue holder, haemostatic forceps, sketch pen tip, ink etc.

EXPERIMENTAL CONDITIONS:

Physiological salt solution :

Temperature :

Basal Tension on lever :

Sensitivity :

Aeration :Contact Time :

PROCEDURE:

The assembly was set up and arrangements were made for experimental conditions

mentioned above.

A rat fasted over night was anaesthetized by chloroform and sacrificed by cutting neck

blood vessels and bleeding the animal to death.

The abdominal cavity was quickly opened through a midline incision, the ileocaecal

junction exposed, the terminal ileum was cut after discarding 10 cm nearest to theileocaecal junction because of the presence of excitatory - adrenoreceptor near the



7/31/2019 MBE PART 1 (1)

10/44


ileocaecal junction. It was placed in a petridish containing tyrode solution maintained at

370C.

The mesenteric attachment was cut as close to the gut as possible without injury for a

distance of about 20-25 cm. The intestine was then cut across, and the lumen of the

isolated piece thoroughly cleaned by running warm salt solution repeatedly through the

proximal opening with the help of 50 ml volumetric (bulb) pipette (held at an angle ofabout 20 30 degrees). Undue stretching, ballooning or handling of the gut was avoided.

The clean strip of the intestine was then placed in fresh warm salt solution for a short

period for acclimatization before being put up. (If strips were to be kept for further use,

they should be better placed directly in ice-cold aerated salt solution and kept in

refrigerator overnight: about two hour before use they should be transferred to salt solution

at room temperature and actively aerated.)

A small segment (2-3 cm, depending on the size of the organ tube) was cut: a thread

was passed through the lumen and the wall near the mesenteric attachment at each end with

the help of a fine curved sewing needle and tied securely but without occluding the lumen.

The tissue was mounted in mammalian organ bath in the up-right position and

connected to isotonic frontal writing lever under a tension of 500 mg. The tissue was

allowed to stabilize for 30 minutes during which period washing was given at an interval of

10 min.

For maximum sensitivity the lever was nearly balanced, and the friction at the writing

surface reduced to a minimum by smooth point.

Starting with the equipotent responses, the responses of acetylcholine were taken till

maximum effect was obtained.

Two equi-active responses of test sample were taken in such a manner that the height

of response lay in between 20% to 80% response of the acetylcholine (It is better to start

with the diluted unknown solution).

The height of contraction was measured and plotted against the log dose. The dose of

standard producing the same response as produced by the test was read directly from the

graph and the concentration of test sample was determined by the formula as mentioned

below.

Dose of STD.

Concentration of unknown = ----------------- X Conc. of STD. x dilution factor

Dose of TEST



7/31/2019 MBE PART 1 (1)

11/44


STANDARD PATTERN:

OTHER METHODS FOR BIOASSAY OF ACETYLCHOLINE:

Apart from using rectus abdominis muscle of frog or rat-ileum one can perform bioassay of

acetylcholine on guinea-pig ileum and leech dorsal muscle. Some laboratories have

reported use of isolated heart, intestine and tracheal preparations. However, overall

experience dose not recommend these method as reliable, reproducible or accurate. Amongwhole animal experiments cat or rat blood pressure experiments have also been suggested.

However, these are tiresome and not commonly used.

Guinea-pigs Ileum: Guinea pig is killed by a below on the head and bled to death. The

abdominal wall is dissected out so as to isolate the ileum; the faecal matter, mesentery and

blood vessels are removed from the piece of ileum.

It is ligated on both sides and suspended in mammalian at 37O C and oxygenated

continuously. Acetylcholine contracts the ileum. This principle is utilized for its bioassay.The extent of contraction produced by the test sample is compared with the standard

preparation of acetylcholine.M. Pharm II-semester (Pharmacology) - 11 -


7/31/2019 MBE PART 1 (1)

12/44


Leech Dorsal Muscle: Compare the contractions produced by the standard and test

samples on eserinised dorsal muscle of the leech. This muscle is highly sensitive

(picograms) to acetylcholine.

Isolated Heart Preparations: Rabbits auricle, frogs heart, rabbits heart or venousmercenerials heart is used. Ach decreases the force of contraction and rate of the heart.

Rabbits Intestine and Tracheal chain: Ach contracts these tissues.

Cats Blood Pressure: A cat is anaesthetized with suitable anesthetic. The carotid artery is

cannulated for recording blood pressure femoral vein is cannulated for injecting

acetylcholine. Trachea is cannulated for giving artificial respiration.

Acetylcholine produces a fall in blood pressure by dilating peripheral blood vessels. This

principle is utilized for its bioassay. The extent to which blood pressure falls due to the test

sample is compared with the fall by the standard preparation.

Anaesthetized Rats Blood Pressure: Compare the extent of fall in blood pressure of the

test sample with that produced by the standard preparation.

CALCULATION & OBSERVATIONS:

Drug:

Stock Concentration:

Bath capacity:

OBSERVATION TABLE:

Sr. No.

Concentration

of the drug

(g/ml)

Dose of Drug

(ml)

Height of

Response of the

drug (mm)

GRAPH:



7/31/2019 MBE PART 1 (1)

13/44

7/31/2019 MBE PART 1 (1)

14/44


The concentration of given unknown sample of the drug - _______________ is

_____________ g/ml.



Teachers sign

7/31/2019 MBE PART 1 (1)

15/44


Date:

PRACTICAL-3

Bioassay of agonist-Acetylcholine by Matching Method using rat ileum preparation

AIM: To find out the concentration of unknown sample of acetylcholine by matching

method using rat ileum.

PRINCIPLE:

Acetylcholine produces a dose dependent contraction of rat ileum smooth muscle. Two

responses of the standard acetylcholine are taken. The does are adjusted such that one is

giving response of approximately 20% and other 70% of the maximum. The response of

unknown that lies between two responses of standard acetylcholine dose is taken. The

panel is repeated by increasing or decreasing the doses of standard till all three equal

responses are obtained. The dose of test sample is kept constant. In the end, a response of

the double dose of the standard and test that match each other are taken. and the

concentration of unknown is determined by the formula.

REQUIREMENTS:




Thermostat, isotonic transducer & Coupler, aeration tube cum Tissue

holder, haemostatic forceps, ink etc.



Temperature :


Sensitivity :

Aeration :

Contact Time :

PROCEDURE: The assembly was set up and arrangements were made for experimental conditions

mentioned above.


blood vessels and bleeding the animal to death. The abdominal cavity was quickly opened

through a midline incision, the ileocaecal junction exposed, the terminal ileum was cut

after discarding 10 cm nearest to the ileocaecal junction because of the presence of

excitatory - adrenoreceptor near the ileocaecal junction. It was placed in a petridish

containing tyrode solution maintained at 370C.



isolated piece thoroughly cleaned by running warm salt solution repeatedly through theM. Pharm II-semester (Pharmacology) - 15 -


7/31/2019 MBE PART 1 (1)

16/44


proximal opening with the help of 50 ml volumetric (bulb) pipette (held at an angle of

about 20 30 degrees). Undue stretching, ballooning or handling of the gut was avoided.




refrigerator overnight: about two hour before use they should be transferred to salt solutionat room temperature and actively aerated.)




The tissue was mounted in mammalian organ bath in the up-right position and connected to

isotonic frontal writing lever under a tension of 500 mg. The tissue was allowed to stabilize

for 30 minutes during which period washing was given at an interval of 10 min.



Starting with the equipotent responses, the responses of acetylcholine were taken till


Initially, two responses of the std. are taken. The doses are adjusted such that one is

giving responses of aprrox.20% and other 70% of the maximum.

The response of unknown which lies between two responses of std dose is taken. The

panel is repeated by increasing or decreasing the dose of std. till all the equal responses are

obtained. The dose of test sample is kept constant.

At the end a response of the double dose of the std & test which match each other are

taken. There should give equal responses.

Then by using the following formula corresponding conc. of test is obtained.

Dose of STD.


Dose of TEST

STANDARD PATTERN:

CALCULATION & OBSERVATIONS



7/31/2019 MBE PART 1 (1)

17/44


Drug:


Bath capacity:

OBSERVATION TABLE:

Sr. No.

Concentration

of the drug

(g/ml)

Dose of Drug

(ml)

Height of

Response of the

drug (mm)

CALCULATION:

Dose of STD.

Concentration of unknown = ----------------- X Conc. of STD. x Dilution FactorDose of TEST

RESULT:


_____________ g/ml.

Date:

PRACTICAL-4Bioassay of agonist-Acetylcholine by Three Point Method using rat ileum preparation



Teachers sign

7/31/2019 MBE PART 1 (1)

18/44


AIM: To perform the bioassay of acetylcholine by three point method using rat ileum.

PRINCIPLE:

Acetylcholine produces contractions of smooth muscle of rat ileum. In 3-point assay

method, 2 doses of the standard acetylcholine (S1, S2) and one dose of the testacetylcholine (T) are used. The test dose is fixed in such a way that it gives the response

between the responses produced by S1 & S2. S1 & S2 are the doses of standard that are

20% & 70% of the maximum response. These three selected doses are repeated by the

Latin Square design method i.e. S1,S2,T S2,T,S1 T,S1,S2. The mean responses are

calculated and plotted against log-dose and amount of standard producing the same

response as produced by the test is determined mathematically.

REQUIREMENTS:








Temperature :

Basal Tension on lever :Sensitivity :

Aeration :

Contact Time :

PROCEDURE:


mentioned above.


blood vessels and bleeding the animal to death. The abdominal cavity was quickly openedthrough a midline incision, the ileocaecal junction exposed, the terminal ileum was cut













7/31/2019 MBE PART 1 (1)

19/44







the help of a fine curved sewing needle and tied securely but without occluding the lumen.The tissue was mounted in mammalian organ bath in the up-right position and connected to





Response of smaller dose of acetylcholine approximately producing 20% of the

response (S1) was taken. Consider the dose as n1.

Response of higher dose of acetylcholine approximately producing 80% of the

response (S2) was taken. Consider the dose as n2. Responses of the test solution were taken in such a way that the height of response

produced lie between the responses produced by n1 and n2 dose. Consider the dose as t.

The sequence of responses was followed as per the Latin square method of

randomization in order to avoid bias. Then responses were taken in pattern of S2-T-S1 and

then T-S1-S2.

The mean responses were calculated and plotted against log dose and amount of

standard producing the same response as produced by the test was determined

mathematically.

n1 T S1 n2

Conc. of Unknown= Cs X ---- X antilog ---------- X log ---- X dilution factor

t S2 S1 n1



t = Test dose

S1 = Response of n1

S2 = Response of n2

T = Response of t

Cs = Conc. of Std.

STANDARD PATTERN:



7/31/2019 MBE PART 1 (1)

20/44



Drug:


Bath capacity:

OBSERVATION TABLE:

Concentration

of the drug

(g/ml)

Dose of Drug

(ml)

Height of Response of the drug (mm)

I II III Mean

S1

S2

T

CALCULATION:

n1 T S1 n2

Conc. of Unknown= Cs X ---- X antilog ---------- X log ---- X Dilution Factor

t S2 S1 n1



S2S1 T S2 S1T S2S1T

7/31/2019 MBE PART 1 (1)

21/44




t = Test dose

S1 = Response of n1

S2 = Response of n2T = Response of t

Cs = Conc. of Std.

RESULT:


_____________ g/ml.



Teachers sign

7/31/2019 MBE PART 1 (1)

22/44


Date:

PRACTICAL-5

Bioassay of agonist-Acetylcholine by Four point method using rat ileum preparation

AIM: To perform the bioassay of acetylcholine by four point method using rat ileum.

PRINCIPLE:

Acetylcholine produces contractions of smooth muscle of rat ileum. In 3-point assay

method, 2 doses of the standard acetylcholine (S1,S2) and one dose of the test

acetylcholine(T) are used. The test dose is fixed in such a way that it gives the response

between the responses produced by S1 & S2. S1 & S2 are the doses of standard that are

20% & 70% of the maximum response. Select two concentrations (A,B) of the standard

drug, eliciting sub maximal responses (S1,S2) and bearing a dose ration 1:2 preferentially.

Select two suitable volumes of the test solution by trial and error method in such a way that

the response (T1) due to the lower dose of the test (C) lies preferentially between S1 & S2.

The higher volume of the test solution selected would be D such that the dose ratio

B/A=D/C. All the four responses (S1,S2,T1,T2) due to the doses thus selected (A,B,C,D)

must lie on the linear part of the standard (sigmoid) curve.

These four selected doses are repeated by the Latin Square design method The mean

responses are calculated and concentration of the test is determined mathematically.

REQUIREMENTS:








Temperature :

Basal Tension on lever :Sensitivity :

Aeration :

Contact Time :

PROCEDURE:


mentioned above.







7/31/2019 MBE PART 1 (1)

23/44







proximal opening with the help of 50 ml volumetric (bulb) pipette (held at an angle ofabout 20 30 degrees). Undue stretching, ballooning or handling of the gut was avoided.














Record graded response with the standard solution of acetylcholine until peak effect is

obtained.

Select two concentrations (A,B) of the standard drug, eliciting sub maximal responses

(S1,S2) and bearing a dose ration 1:2 preferentially.

Select two suitable volumes of the test solution by trial and error method in such a way

that the response (T1) due to the lower dose of the test (C) lies preferentially between S1 &

S2. The higher volume of the test solution selected would be D such that the dose ratio

B/A=D/C. All the four responses (S1, S2, T1, T2) due to the doses thus selected (A, B, C,

D) must lie oh the linear part of the standard (sigmoid) curve.

Standardize the tissue with concentration A. (Tissue is said to be standardized when it

responds identically to the same concentration, when repeated).

The sequence of responses was followed as per the Latin square method of

randomization in order to avoid bias. Record four sets of responses due to A,B,C,D adding

them to the organ bath in a randomized fashion. Any of the following latin squares may beused to ensure good randomization and to account for the fluctuating sensitivity of the

tissue.

STANDARDIZATION AND FOUR CYCLES USING LATIN SQUARE DESIGN

ABCD ABCD ABCD ABCD

BCDA BADC BDAC BADC

CDAB CDBA CADB CDAB

DABC DCAB DCBA DCBA

Measure various responses were measure to calculate the mean of each response (S1,

S2, T1, and T2).

Calculate the potency ratio (M) using formula:



7/31/2019 MBE PART 1 (1)

24/44


(T2-S2) + (T1-S1)

Potency ratio = M = (x1/y1) X antilog -------------------- X log (x2/x1)

(T2-T1) + (S2-S1)

Where,

x1 = Lower volume of std. Drug(A)x2 = Higher volume of std. Drug(B)

y1 = Lower volume of test drug(C)

S & T = Represent mean response

Determine the strength of unknown solution of acetylcholine using the concentration of

the standard (1 mg/ml), dilution factor for the test solution and the potency ratio (M).

STANDARD PATTERN:

A B C D AB C D

A BC D A B CD

S1

S2

T1

T2

S1

S1 S1

S2

S2 S2

T1

T1 T1

T2

T2 T2


Drug:


Bath capacity:

OBSERVATION TABLE:



7/31/2019 MBE PART 1 (1)

25/44


Concentration

of the drug

(g/ml)

Dose

of

Drug

(ml)

Height of Response of the drug (mm)

I II III IIII Mean

S1

S2

T1

T2

CALCULATION:

(T2-S2) + (T1-S1)

Potency Ratio= M =(x1/y1) X antilog ------------------- X log(x2/x1)

(T2-T1) + (S2-S1)

Where,

x1 = Lower volume of std. Drug(A)

x2 = Higher volume of std. Drug(B)

y1 = Lower volume of test drug(C)

S & T = Represent mean response

RESULT:

The concentration of given unknown sample of the drug - _______________ is_____________ g/ml.



Teachers sign

7/31/2019 MBE PART 1 (1)

26/44


Date:

PRACTICAL-6

Bioassay of antagonist-Atropine by Graphical Method using rat ileum preparation

AIM: To find out the concentration of unknown sample of atropine by graphical method

using rat ileum.

PRINCIPLE:

Acetylcholine produces contractile responses on rat ileum through muscarinic receptors.

Atropine is a muscarinic receptor blocker. Atropine has a very slow dissociation rate. Thus

recovery is slow. One has to wait for a longer period of 10-15 min for the recovery to come.

Also large dose of Ach would not speed up the removal of atropine. A blocking agent

produces dose dependent competitive and reversible antagonist of acetylcholine. Hence

graded responses of atropine in the form of inhibition of the fixed dose of acetylcholine can

be determined. The percent inhibition is plotted against log dose of atropine and the

concentration of unknown is determined by finding out the amount of standard producing

same response (inhibition) as produced by unknown.

REQUIREMENTS:


Drugs : Acetylcholine (10 g/ml, 100 g/ml, 1 mg/ml),

Atropine (1 g/ml, 10 g/ml, 100 g/ml)






Temperature :


Sensitivity :

Aeration :

Contact Time :

PROCEDURE:


mentioned above.





excitatory - adrenoreceptor near the ileocaecal junction. It was placed in a petridishcontaining tyrode solution maintained at 370C.



7/31/2019 MBE PART 1 (1)

27/44







The clean strip of the intestine was then placed in fresh warm salt solution for a shortperiod for acclimatization before being put up. (If strips were to be kept for further use,












Two equipotent responses to sub maximal doses of acetylcholine were recorded. The

drum was moved for 30 seconds and the lowest dose of Atropine was added in bath. After

2 minutes, responses to the same dose of acetylcholine were taken in the presence of

atropine. The 5-minute cycle was followed as usual.

The responses to acetylcholine were taken after every 5 minutes till the recovery to the

control height was achieved. The response to acetylcholine in presence of higher dose of

atropine was taken. At least 4 such dose dependent inhibitions were recorded. In case the

recovery was not achieved even after repeated doses then either a large dose of

acetylcholine was given for recovery or the preceding height was taken as control for the

next dose.

In the same fashion the responses to acetylcholine were produced in presence of

unknown solution of atropine at least twice.

Heights of control (s = in absence of atropine) and test (t = in presence of atropine)

were measured and the % inhibition was calculated as follows:

control test% Inhibition = ------------------ X 100

control

The percent inhibition was plotted on log graph and the concentration of unknown was

then calculated out.

Dose of STD.

Concentration of unknown = ----------------- X Conc. of STD. x Dilution Factor

Dose of TESTCALCULATION & OBSERVATIONS:



7/31/2019 MBE PART 1 (1)

28/44


Drug:

Agonist

Antagonist


Agonist

AntagonistBath capacity:

OBSERVATION TABLE:

Sr.

No.

Concentration of

the antagonist

(g/ml)

Dose of

antagonist

(ml)

Height of

response of

agonist in absence

of antagonist

(control response

C mm)

Height of

response of

agonist in

presence of

antagonist (Test

response T

mm)

Percentage

inhibition of

control

response

C-T x 100

C

(%)

GRAPH:

Plot Graph: Percentage inhibition of the control response (Y-axis) vs.

The Log dose of antagonist (X-axis)

Height of contraction of test drug on y-axis = dose on x-axis (taken as dose of standard in

the formula)

CALCULATION:

Dose of STD.


Dose of TEST

RESULT:


_____________ g/ml.

Date:PRACTICAL-7



Teachers sign

7/31/2019 MBE PART 1 (1)

29/44


Bioassay of agonist-Histamine by Graphical Method using rat fundus preparation

AIM: To find out the concentration of unknown sample of histamine by graphical method

using rat fundus strip

PRINCIPLE:This method is based on the assumption of the dose-response relationship.

Histamine produces a dose dependent contraction of rat fundus smooth muscle. Graded

responses of acetylcholine are taken and two equipotent responses to unknown sample are

taken. In simpler design, 5-6 response of the graded doses of the standard are taken and

then two equi-active response of the test sample are taken. The height of the contraction is

measured and Log-dose-response curve is plotted. The dose of standard producing the

same response as produced by the test sample is directly read from the graph and the


Advantages:

- Most simple method.

- Chances of error are less if the sensitivity of the preparation is not changed.

REQUIREMENTS:


Drugs : Histamine (10 g/ml, 100 g/ml, 1 mg/ml)






Temperature :


Sensitivity :

Aeration :

Contact Time :

PROCEDURE:


mentioned above.



through a midline incision, and the stomach dissected out and placed in warm salt solution.

The translucent fundus (rumen) was cut along the pylorus (thick and red ) leaving a thin

band of the pyloric tissue attached to the fundus and its contents were washed clean.

The fundus was then cut open along the lesser curvature and spread on a cork mat

soaked in salt solution. Alternative transverse cuts were then made to preserve the

longitudinal muscle. The strip was then pulled out by cotton thread tied on each end andM. Pharm II-semester (Pharmacology) - 29 -


7/31/2019 MBE PART 1 (1)

30/44


protrusion and fringes of the pyloric tissue trimmed away to give a long clean thin strip for

suspension in the bath.




10 min. For maximum sensitivity the lever was nearly balanced, and the friction at the writing


Starting with the equipotent responses, the responses of histamine were taken till



of response lay in between 20% to 80% response of the histamine (It is better to start with

the diluted unknown solution).


standard producing the same response as produced by the test was read directly from thegraph and the concentration of test sample was determined by the formula as mentioned

below.

Dose of STD.


Dose of TEST

RESULT:The concentration of given unknown sample of the drug- _______________ is

_____________ g/ml.

DISSCUSSION :

It is suitable for assay of 5-Hydroxytryptamine, being very sensitive to its

action. It is 10 times less sensitive to acetylcholine, the effect of which can be

blocked by hyoscine, while it is over 1000 times less sensitive to histamine. It is

also used for the assay of PGE2.



Teachers sign

7/31/2019 MBE PART 1 (1)

31/44


Date:

PRACTICAL-7

Bioassay of agonist-histamine by Graphical Method using rat fundus preparation


Drug:Stock Concentration:

Bath capacity:

OBSERVATION TABLE:

Sr.

No.

Concentration

of the drug

(g/ml)

Dose of Drug

(ml)

Log dose of

drug

Height of

Response of the

drug (mm)

Percentage

height of

contraction (%)

GRAPH:

Plot Graph: Height of contraction (Y-axis) vs. the Log dose of drug (X-axis)


the formula)

CALCULATION:

Dose of STD.


Dose of TEST



7/31/2019 MBE PART 1 (1)

32/44


Date:

PRACTICAL-8

Bioassay of agonist-Serotonin by Graphical Method using rat fundus preparation

AIM: To find out the concentration of unknown sample of Serotonin by graphical method

using rat fundus strip

PRINCIPLE:

This method is based on the assumption of the dose-response relationship. Serotonin

produces a dose dependent contraction of rat fundus smooth muscle. Graded responses of

acetylcholine are taken and two equipotent responses to unknown sample are taken. In

simpler design, 5-6 response of the graded doses of the standard are taken and then two

equi-active response of the test sample are taken. The height of the contraction is measured

and Log-dose-response curve is plotted. The dose of standard producing the same response

as produced by the test sample is directly read from the graph and the concentration of

unknown is determined by the formula.

Advantages:

- Most simple method.

- Chances of error are less if the sensitivity of the preparation is not changed.

REQUIREMENTS:


Drugs : Serotonin (10 g/ml, 100 g/ml, 1 mg/ml)

Apparatus : Reservoir, tubing, Mammalian isolated organ bath, organ tube,

heating coil, Thermostat, isotonic frontal writing lever, recording

drum, aeration tube cum Tissue holder, haemostatic forceps, sketch

pen tip, ink etc.



Temperature :


Sensitivity :


PROCEDURE:


mentioned above.



through a midline incision, and the stomach dissected out and placed in warm salt solution.

The translucent fundus (rumen) was cut along the pylorus (thick and red ) leaving a thinband of the pyloric tissue attached to the fundus and its contents were washed clean.



7/31/2019 MBE PART 1 (1)

33/44

7/31/2019 MBE PART 1 (1)

34/44


Date:

PRACTICAL-8

Bioassay of agonist-serotonin by Graphical Method using rat fundus preparation



Bath capacity:

OBSERVATION TABLE:

Sr.

No.

Concentration

of the drug

(g/ml)

Dose of Drug

(ml)

Log dose of

drug

Height of

Response of the

drug (mm)

Percentage

height of

contraction (%)

GRAPH:



the formula)

CALCULATION:

Dose of STD.


Dose of TEST

Date:

PRACTICAL-9



7/31/2019 MBE PART 1 (1)

35/44


Bioassay of agonist- Dopamine by Graphical Method using rat vas deferens

preparation

AIM: To find out the concentration of unknown sample of Dopamine by graphical

method using rat vas deference.

PRINCIPLE:

This method is based on the assumption of the dose-response relationship. Dopamine

produces a dose dependent contraction of rat vas deferens smooth muscle. Graded







ADVANTAGES:

Most simple method.


REQUIREMENTS:


Drugs : Dopamine (10 g/ml, 100 g/ml, 1 mg/ml)




pen tip, ink etc.



Temperature :


Sensitivity :


PROCEDURE:


mentioned above.



through a midline incision, and two vas deference dissected out and placed in warm salt

solution.



7/31/2019 MBE PART 1 (1)

36/44





10 min.


surface reduced to a minimum by smooth point. Starting with the equipotent responses, the responses of Dopamine were taken till








below.Dose of STD.


Dose of TEST

RESULT:


_____________ g/ml.

DISSCUSSION :

In comparison with other smooth muscle like aorta, trachea, vas-deference is more

easy to dissect. As it is available in pairs the control and the test preparation can be

done from the same animal. It is good preparation to study sympathetic nerve trunk

and its relations with drug.

The vas-deference arises from the caudal epididymis which is situated at the

posterior end of testes, leads back through the inguinal canal and crosses the ureter

before joining the urethra. They are 5 to 7 cm in length covered by a thin layer of

connective tissues and surrounded by fat cells. The vas-deference of rat is supplied

with hypo gastric nerve.

The muscle contains dense plexus of catecholamine neurons as adrenergic nerves

extensively innervate it.

It has been reported that simply stripping away the serous coat after removing the

vas-deference from the animal increases the sensitivity of the vas deference to drugs

by facilitating access of drugs to the smooth muscle cells. Thus some of the increase

in sensitivity to adrenaline and nor adrenaline

found after denervation can be attributed to this

basis.



Teachers sign

7/31/2019 MBE PART 1 (1)

37/44


Date:

PRACTICAL-9

Bioassay of agonist- Dopamine by Graphical Method using rat vas deferens



Bath capacity:

OBSERVATION TABLE:

Sr.

No.

Concentration

of the drug

(g/ml)

Dose of Drug

(ml)

Log dose of

drug

Height of

Response of the

drug (mm)

Percentage

height of

contraction (%)

GRAPH:



the formula)

CALCULATION:

Dose of STD.


Dose of TEST

Date:

PRACTICAL-10



7/31/2019 MBE PART 1 (1)

38/44


Bioassay of agonist- Oxytocin by Graphical Method using rat uterine horns

AIM: To find out the concentration of unknown sample of Oxytocinn by graphical

method using rat uterine horns

PRINCIPLE:This method is based on the assumption of the dose-response relationship. Oxytocin

produces a dose dependent contraction of rat uterine horns smooth muscle. Graded







ADVANTAGES:

Most simple method.


REQUIREMENTS:


Drugs : Oxytocin (1-100 IU)




pen tip, ink etc.



Temperature :


Sensitivity :

Aeration :

Contact Time :

PROCEDURE:


mentioned above.

A virgin female rat was injected 100 g/100 gms body weight of Diethylstilbestrol, 24

hours before it was sacrificed. The assembly was set up and arrangements were made for

experimental conditions mentioned above.


blood vessels and bleeding the animal to death The abdominal cavity was quickly



7/31/2019 MBE PART 1 (1)

39/44


opened through a midline incision, and the uterine horns were dissected out.

They was placed in a petridish containing de-Jalon solution maintained at 370C.

The entire uterine horn was mounted in mammalian organ bath in the up-right

position and connected to isotonic frontal writing lever under a tension of 0.5-1 g. The

tissue was allowed to stabilize for 30 minutes during which period washing was given at an

interval of 10 min. For maximum sensitivity the lever was nearly balanced, and the friction at the writing


Starting with the equipotent responses, the responses of Oxytocin were taken till






standard producing the same response as produced by the test was read directly from thegraph and the concentration of test sample was determined by the formula as mentioned

below.

Dose of STD.


Dose of TEST

RESULT:


_____________ g/ml.

DISSCUSSION :

The rat uterus is chiefly used for the assay of Oxytocin, 5-hydroxytryptamine and

adrenaline. On this tissue Acetylcholine, 5-Hydroxytryptamine, Oxytocin , Barium

Chloride and Potassium chloride produces a contractile effect (spasmogenic effect ).

Hence, such drugs are known as Spasmogenics. While histamine and adrenaline

decreases responses of Potassium chloride and also relax the preparation previously

contracted with a spsasmogen. Such drugs are known as Spasmolytics. Generallyblockers act as spasmolytics but it is interesting to note spasmolytic property of

histamine and adrenaline.

Excitatory -adrenoreceptors have been shown to exist in the rat uterus only under

certain conditions such as after estrogen treatment, during natural oestrus, in the late

pregnancy, and for five to six days after partuation. The adrenoreceptors are

temperature sensitive, and the initial excitation phase produced by several

sympathomimetic amines is greatly reduced or even abolished by lowering the bath

temperature to 25C. For the estimation of oxytocic activity , female rats are keptseparated from males because pregnant animals are not suitable.



7/31/2019 MBE PART 1 (1)

40/44


The rhythmic contractions normally present are abolished by using de-Jalons

solution . To have relatively quiescent but sensitive uterus for routine assays, virgin

rats are injected with stilbesterol 24 hours before sacrificing. For 5-hydroy

tryptamine assay a relatively large dose of stilbestrol (0.25 mg/ 100 gm) is injected

intra-peritoneally for three days before sacrificing.

Histamine by acting on H2 receptors releases nor adrenaline which in turn relaxes

uterine muscles. It is very sensitive to stimulation by posterior pituitary extract ,

bradykinin, substance P and adenosine compounds, and to inhibition of adrenaline

and noradrenaline.

Rats in natural oestrus may also be selected by microscopic examination of the

vaginal smear. The uterus is generally still suitable when kept at 4C for 24 hours.



Teachers sign

7/31/2019 MBE PART 1 (1)

41/44


Date:

PRACTICAL-10

Bioassay of agonist- Oxytocinby Graphical Method using rat uterine horns



Bath capacity:

OBSERVATION TABLE:

Sr.

No.

Concentration

of the drug

(g/ml)

Dose of Drug

(ml)

Log dose of

drug

Height of

Response of the

drug (mm)

Percentage

height of

contraction (%)

GRAPH:



the formula)

Calculation:

Dose of STD.


Dose of TEST

Date:



7/31/2019 MBE PART 1 (1)

42/44


PRACTICAL-11

Bioassay of agonist- Acetylcholine by Graphical Method using rat tracheal chains

AIM: To find out the concentration of unknown sample of acetylcholine by graphical

method using rat tracheal chain.

PRINCIPLE:

Acetylcholine produces a dose dependent contraction of rat trachea smooth muscle. Graded


taken. The responses can be plotted against log dose of standard and the amount of

standard, producing the same response as produced by unknown, is directly read from the

graph and the concentration of unknown is determined by the formula.

REQUIREMENTS:






pen tip, ink etc.



Temperature :


Sensitivity :

Aeration :

Contact Time :

PROCEDURE:


mentioned above.

A rat fasted over night was anaesthetized by chloroform and sacrificed by cutting neckblood vessels and bleeding the animal to death. The neck portion opened through a midline

incision, the trachea dissected out. It was placed in a petridish containing Krebss solution

maintained at 370C.

A transverse cut between the segment of cartilage is given to get a number of rings of

tracheal chain. Rings are tied together with cotton thread to form a chain. Preparation can

also obtained by cutting tracheal chain into zigzag fashion.

The entire tracheal chain was mounted in mammalian organ bath in the up-right

position and connected to isotonic frontal writing lever under a tension of 0.5-1 g. The

tissue was allowed to stabilize for 30 minutes during which period washing was given at an

interval of 10 min.



7/31/2019 MBE PART 1 (1)

43/44




Starting with the equipotent responses, the responses of Acetylcholine were taken till



of response lay in between 20% to 80% response of the acetylcholine (It is better to startwith the diluted unknown solution).




below.

Dose of STD.


Dose of TEST

RESULT:

The concentration of given unknown sample of the drug _______________is

_____________ g/ml.



Teachers sign

7/31/2019 MBE PART 1 (1)

44/44


Date:

PRACTICAL-11

Bioassay of agonist- Acetylcholine by Graphical Method using rat tracheal chains



Bath capacity:

OBSERVATION TABLE:

Sr.

No.

Concentration

of the drug

(g/ml)

Dose of Drug

(ml)

Log dose of

drug

Height of

Response of the

drug (mm)

Percentage

height of

contraction (%)

GRAPH:



the formula)

CALCULATION:

Dose of STD.


Dose of TEST

mbe part 1 (1)

Documents