A bioassay is an analytical method to determine the concentration or potency of a substance by its effect on living animals or plants (in vivo), or on living cells or tissues (in vitro). A bioassay can be either quantal or quantitative, direct or indirect. If the measured response is binary, the assay is quantal, if not, it is quantitative.
In vivo bioassays involve administering the test substance to a living organism to determine the substances pharmacological activity. Various behavioral and physiological parameters are measured before and after administration, allowing the type and mode of pharmacological activity to be adduced. The most important in vivo bioassays for pharmaceutical research are human clinical trials, which are almost always administered after reasonable expectations of safety and efficacy have been determined from animal trials. The power of in vivo assays is their ability to reveal unexpected or novel modes of pharmacological activity since the impact of the test substance on the whole organism can be studied.
INDICATIONS OF BIOASSAY
- Active principle unknown.
- Active principle cannot be isolated.
- To study biological response of new drug.
- To ensure purity & potency.
- If chemical assay not available/ complex/insensitive to low doses
- To estimate concentration of endogenous mediators
PRINCIPLE of bioassays
·
Compare potency of unknown substance with
standard (including assessment of errors).
·
All bioassays (lab
studies, toxicity studies, clinical studies) must be comparative & compared
against a standard drug or preparation. The potency of biologically assayed
compounds is compared with the activity of internationally accepted standards.
·
The standard and
new drugs should be as far as possible identical to each other, then their dose
response curve (DRC) will have the same stop and would be constant all along
the response level.
·
The biological
response should be closely related to the therapeutic use of the drug as
possible. For example, in estimating the potency of insulin, blood glucose
levels would be of the ideal index, but the method commonly used is the mouse
convulsion method, in which the % of animals developing convulsions are
recorded. Similarly for estimating digitaloid drug, the arrest of heart in
pigeon or guinea pig is used as end point which is as the toxic effect of the
drug and not the therapeutic effect.
·
An estimate of
relative potency is made by comparing the log dose of the test and the log dose
of the standard that produces an equal magnitude of effect.
·
The problem of
biological variation must be minimized as far as possible. For that one should
keep uniform experimental conditions and assure the reproducibility of the
responses.
·
The results of the
test should be subjected to statistical analysis to minimize errors due to
biological variations.
·
Standard & test sample should have
same pharmacological effect & mode of action.
·
The test and standard should be compared using
a specified pharmacological technique.
·
Bioassays should be reproducible, and in practice should
be reproduced several times (i.e. with different batches of inoculums,
beginning on different days) to document the extent to which results are consistent.
·
Bioassays
should be conducted using test insects that are of standardized condition
(e.g., of the same instars and not infected by another pathogen, unless this is
part of the study).
·
Bioassays
should be conducted using inoculums that are also of standard characteristics
across the different repetitions of the study.
·
Methods,
conditions and processing used during the bioassay must be consistent for all
replications.
·
To
document that the treatment caused resulting effects, negative control insects
(not treated with the pathogen but receiving similar conditions) must be
included for each replicate of the study.
·
In
some cases, as when testing the survival of the pathogen under different
conditions, positive controls should also be included (test insects treated
with a pathogen and held under conditions known to result in successful
infection).
TYPES
OF BIOASSAY
1.
GRADED BIOASSAY:
It is a response is
proportional to the dose and response may lie between no response and the
maximum response
METHODOLOGY:
·
Checking
of apparatus for normal functioning.
·
preparing
physiological salt solution.
METHODOLOGY:
· Checking of apparatus for normal functioning.
· preparing physiological salt solution.
· Arranging instruments and adjusting water bath and balancing the lever.
· Selecting the tissue (surgical process and selecting the required tissue).
· Tissue attachment to the apparatus setup. Stabilizing the tissue.
· Preparation of the drugs ( standard and test). Fixing the dose.
· Preparing DRC for standard and test drug. selecting the assay for drug and calculations are reported.
·
Preparing
DRC for standard and test drug. selecting the assay for drug and calculations
are reported.
Graded Bioassay
Intermittent dose method:
Intermittent drug dosing intervals are usually initially guided by the
terminal pharmacokinetic half life and are dependent on drug formulation. For
chronic multiple dosing and for extended release dosage forms, the terminal
half life often does not predict the plasma drug accumulation or fluctuation
observed.
|
t1/2,abs |
Absorption
half life |
|
t1/2,β |
Beta pharmacokinetic half life |
|
t1/2,term |
Terminal half life: the longer
half life between the absorption and beta half lives |
|
t1/2,op Cmax |
Operational multiple dosing half
life to two-fold accumulation in Cmax; calculated as the dosing
interval to two-fold accumulation in Cmax |
|
t1/2,op AUC |
Operational multiple dosing half
life to two-fold accumulation in AUC0→Ï„; calculated as the dosing
interval to two-fold accumulation in AUC0→Ï„ |
|
t1/2,op fluct |
Operational multiple dosing half
life to two-fold fluctuation at chronic multiple dosing; calculated as the
dosing interval to a Cmax/Cmin ratio of 2 during
chronic multiple dosing |
Cumulative dose method: In medicine, the total amount of a drug or radiation given to a patient over time; for example, the total dose of radiation given in a series of radiation treatments.
A) Matching Assay: In this type of assay the test substance and the standard are applied and the responses obtained are matched by a trial and error process until they produce equal effects. It is also called as the analytical dilution assay as the assay involves the determination of the factor by which the test substance is either diluted or concentrated in order to produce a response that is equal to that of a known amount of the standard drug A corresponding concentration of the test substance is then calculated.
This assay is generally employed
when the ample amount of sample is available Since the assay does not involve
the recording of CRC, the sensitivity of the preparation is not taken into
consideration.
- The assay does not depend on the assumption of a dose
response relationship.
Disadvantages
- Purely subjective method
- Inefficient as
preliminary effects are not utilized in final assessment
- Lot of experimental errors, which cannot be determined.
- A crude method and not the exact method of determining the potency of a drug
- Precision and reliability are poor
B)
Bracketing bioassay:
Bracketing
bioassay is performed by selecting two standard doses, which will give a close
bracket on either side of the response produced by the unknown. The working
dose of standard is first determined in the sensitive part of dose-response curve,
that is, a dose that will approximately produce 50% of the maximal
concentration. The dose of the standard drug is kept constant throughout the
experiment, in order to have some idea about the change in the sensitivity of
tissue with time.
The standard drug is added at fixed intervals but alternating with the
test so that each response produced by a dose of test substance is bracketed by
responses produced by the dose of standard.
The response of test substance is bracketed between two responses of the
standard. Close bracketing gives more accurate results.
c) Interpolation
Method
This is a simplest form of graded
response assay and involves no statistical data and many calculations. In this
assay the dose response curve is fist obtained from different doses of standard
ach solution. The concentration of unknown is then read from the standard
graph.
Interpolation method of bioassay is less time consuming and yet reliable compare to matching type of bioassay. One of the main advantages of this essay is that the sensitivity of the tissue is first determined by prior plotting of a dose response curve with a known agonist as in the case with acetylcholine. If the linearity of curve is good, one can do very accurate estimate of the test substance unknown sample.
D)
Multipoint assay
a. Three Point Bioassay
In three point bioassay, the DRC
of standard & test samples is first obtained from the responses due to
graded doses. From the DRC of standard, two standard doses are selected in such
a way that they have produced 25% & 50% of the maximal response
respectively & are designated as S1 & S2. The responses of these doses
lie on the steepest & straightest part (linear) of the curve. From the DRC
of test sample one test is selected such that it gives a response which lies in
between the two standard responses that is it gives a greater response than S1
& a smaller response than S2 & is designated as T.
After selecting the standard &
test doses, the bioassay is performed by recording the standard & test
responses in randomized fashion as per Latin square design. The pattern of addition
of doses is S1, S2, T; S2, T, S1 & T, S1, S2 in 3 successive cycles. The
mean values of height of contraction for all the 3 doses are calculated and are
used in plotting the graph so as to estimate the potency of the test sample.
The precision and
reliability of this method is much better than matching and bracketing methods
of bioassay & the sensitivity of the isolated tissue preparation is
assessed prior to testing the unknown sample.
Procedure
[Eg Ach bioassay]
Log dose response
curve plotted with varying conc of std Ach solutions and given test solution
Select two std
doses s1& s2 [in 1:2 dose ratio] from linear part of LDR [ Let the
corresponding response be S1, S2]
Choose a test dose
t with a response T between S1 & S2
Record 4 sets data
[Latin square: Randomisation reduces error] as follows
Plot mean of S1,
S2 and T against dose. Calculate
OR
Log Potency ratio
[M] = [(T-S1)/(S2-S1) ] X log d [d= dose ratio]
Advantages: Quick and with more precision than a matching assay. There is a
possibility of using certain statistical procedures although not with much
confidence.
Disadvantages: still an inherent lack of precision, no accuracy
b.
Four Point Bioassay
The classic 2X2 parallel assay
involves being able to measure parallelism where drugs acting through the same
mechanism are expected to produce parallel dose-response curves.
Procedure [Eg Ach bioassay]
Log dose response curve plotted
with varying conc of std Ach solutions and given test solution
Select two std doses s1& s2
from linear part of LDR [ Let the corresponding response be S1, S2]
Choose two test doses t1 & t2
with response T1 &T2 between S1 & S2; Also s2/s1 = t2/t1 = 2
Record 4 data sets [Latin square: Randomization reduces error]
Plot mean of S1, S2 and T1, T2 against dose. Calculate
Log Potency ratio [M] = [(T1-S1+T2-S2) / (S2-S1+T2-T1)] X log d [d = dose ratio]
1. Quantal bioassay
Quantal
means the response is in the form of "all or none", i.e. either maximum
response or no response. In this type of bioassay, there is no intermediate
response. For example animal receiving a convulsant drug either show
convulsions or does not. Examples of drugs assayed by quantal bioassay method
are:
Digitalis: by cardiac arrest in guinea pig or pigeon.
d-Tubocurarine: by rabbit head drop method.
Insulin: by hypoglycaemic convulsions produced in mice.
Though the method is not very accurate, it is employed for comparison of
threshold response and comparison of ED50 or LD50 values.
The
quantal bioassay is an end point method.
a) End point method: Here the threshold dose showing a positive effect
is measured on each animal and the comparisons between two groups of animals
are done i.e. one receiving standard and other the test compounds. ‘
e.g. Bioassay of digitalis in cats. Here the
cat is anaesthetized with chloralose and its 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.
Two groups of animals, one using standard digitalis and the other using test
preparation of digitalis and then potency is calculated as follows:
Concentration of unknown =
Threshold
dose of standard Threshold dose of test ×
Concentration of standard
BIOASSAY OF INSULIN:
·
Insulin is a hormone made by
pancreatic β cell. Insulin is synthesised as pro-insulin in the endoplasmic
reticulum and is processed to the biologically active form inside the
secretory granules.
·
Bioassay of insulin is generally carried out by rabbit method and mice
method.
·
Some of the other methods are: Rat diaphragm method, Epididymal fat pad
of rats,
·
Standard preparation and unit: It is pure, dry and crystalline insulin.
One unit contains 0.04082 mg. This unit is specified by Ministry of Health,
Government
of India and is equivalent to international unit.
·
Preparation of standard solution: Accurately weigh 20 units of insulin
and dissolve it in normal saline. Acidify it with HCl to pH 2.5. Add 0.5% phenol
as preservative.
Add 1.4% to 1.8% glycerine. Final volume should contain 20units/ml. Store the
solution in a cool place and use it within six months.
·
Preparation of test sample solution: The solution of the test sample is
prepared in the same way as the standard solution.
1) Rabbit method:
·
Selection of rabbits: They
should be healthy, weighing about 1800-3000 gms. They should then be maintained
on uniform diet but are fasted for 18 hrs. before assay.
Water is withdrawn during the experiment.
·
Standard and Sample Dilutions: These
are freshly prepared by diluting with normal NaCl solution so as to contain 1
unit/ml and 2 units/ml.
·
Doses: The dose which
can produce suitable fall in blood sugar level is calculated for the standard.
·
Principle: The potency of a
test sample is estimated by comparing the hypoglycemic effect of the sample
with that of the std. preparation of insulin. Any other suitable
method can also be used.
·
Experimental Procedure:
Animals are divided into 4 groups of 3 rabbits each. The rabbits are then put
into an animal holder. They should be handled with care to avoid
excitement.
·
First part of the Test: A
sample of blood is taken from the marginal ear vein of each
rabbit. Presence of reducing sugar is estimated per 100 ml of blood by a suitable
chemical method. This concentration is called‘Initial Blood Sugar Level’.
·
The four groups of rabbits are then given
sc. injections of insulin as follows:
From each rabbit, a sample of
blood is withdrawn up to 5 hrs. at the interval of 1 hr. each. Blood sugar is
determined again. This is known as ‘Final Blood Sugar Level’.
·
Second
part of the test (Cross over test) : The same animals are used for the second part. The
experiment can be carried out after one week. Again they are fasted and initial
blood sugar is determined. The grouping is reversed, that is to say, those
animals which received the standard are given the test and those which received
the test are now given the standard. Those animals which received the less dose
of the standard are given the higher dose of the test sample and vice-versa.
This test is known as ‘Twin Cross Over Test’.
·
Mean
percentage of decrease in blood sugar of first part and second part is
calculated.
BIOASSAY
OF OXYTOCIN:
Oxytocin is a peptide hormones and Neuropeptide. Oxytocin is normally produced
by the paraventricular nucleus of the hypothalamus and released by the
posterior pituitary.
Oxytocin
is a natural hormone that causes the uterus to contract. Oxytocin is used to
induce labour or strengthen labour contractions during childbirth, and to
control bleeding after childbirth.
Oxytocin
is also used to stimulate uterine contractions in a woman with an incomplete or
threatened miscarriage.
Different
bioassay methods used for oxytocin are
·
By contraction of the rat uterus.
·
By depression of the blood pressure in
chicken
·
By measurement of milk ejection pressure
in a lactating rat etc.
Principle:
·
The potency of oxytocin injection is
determined by comparing its activity with that of the standard preparation of oxytocin
under the conditions of the following method of assay.
Standard
preparation:
·
The standard preparation is consisting of
a freeze dried preparation of oxytocin with human albumin and citric acid
(supplied in ampoules containing 12.5 units) or any other suitable preparation,
the potency of which had been determined in relation to the International
standard.
·
The Unit is the specific oxytocin activity
corresponding to that yielded by 0.0005 g of the Standard preparation.
·
The standard preparation is as per the 4th
international standard for Oxytocin, established in 1978.
·
Experimental Methods:
1) By contraction of the rat uterus:
·
Use
female rats weighing between 120 and 200 g. Immediately before the assay
confirm the rat is in oestrous or proestrus by vaginal smear.
·
Inject 100 microgram of oestradiol
benzoate intramuscularly 18-24 hours before the assay.
·
Sacrifice the rat and suspend one horn of
the uterus in a bath containing a solution of the following composition.
Composition (% w/v)
Sodium
chloride
0.662
Potassium chloride
0.045
Calcium chloride 0.007
Sodium bicarbonate
0.256
Disodium hydrogen phosphate 0.029
Sodium dihydrogen phosphate 0.003
Magnesium chloride 0.010
Dextrose
0.050
·
Maintain the bath at a temperature of 32º
C so that spontaneous contraction of the uterus are abolished and preparation
maintain its sensitivity.
·
Oxygenate the solution with a mixture of
95% of oxygen and 5% of carbon dioxide Record the contractions of
the muscles produced by the addition to the bath of two doses of the Standard
Preparation suitably diluted with the above solution.
·
The doses should be such as to produce clearly discriminated, submaximal
contractions. The required doses normally lie between 10 and 50 micro Unitsper
ml of bath liquid.
·
When maximal contraction has reached replace the bath liquid by a fresh
solution.
·
The doses should be added at regular intervals of 3 to 5minutes
depending upon the rate of recovery of the muscle.
·
Dilute the preparation being examined so as to obtain the responses on the
addition of two doses similar to those obtained with the Standard preparation.
·
The ratio between the two doses of the preparation being examined should
be the same as that between the two doses of the Standard Preparation and this
ratio should kept constant throughout the assay.
·
The two doses of Standard preparation and the two doses of the test
preparation should be given according to a randomized order or Latin square
design and at least six responses to each should be recorded.
·
Measure all the responses and calculate the result of the assay by
statistical methods.
2) By
depression of the blood pressure in chicken
·
Anaesthetize a young healthy adult cockerel weighing 1.2 to 2.3 kg with
an anaesthetic that will maintain a prolonged and constant high blood pressure.
·
Expose the gluteus primus muscle in one thigh and cut and retract it to
reveal the popliteal artery and crura vein.
·
Cannulate the popliteal artery and record the blood pressure. Cannulate
the crural or brachial vein.
·
Prepare standard solution with saline so that the volume to be injected
is between 0.1- 0.5 ml.
·
Inject 2 doses of standard solution into cannulated vein and record
blood pressure. The doses should be such that as to produce clearly
discriminated, precipitous, submaximal decreases in B.P.
·
The required doses normally lie between 20 and 100 milli units.
·
The interval between injections should be constant and lie between 3-10
minutes depending on the rate at which B.P. returns to normal.
·
Dilute test sample before use with saline solution so as to obtain
responses similar to those obtained with the standard preparation.
·
The ratio between the two doses of the
test preparation being examined should be same as that between the two doses of
the standard preparation and the ratio should be kept constant throughout the
assay.
·
The two doses of Standard preparation and
the two doses of the test preparation should be given according to a randomized
order or Latin square design and at least six responses to each should be
recorded
·
If
animal rapidly become insensitive due to repeated injections of the solutions
another animal must be used. Measure all responses and calculate the result by
standard statistical methods
3) By
measurement of milk-ejection pressure in a lactating rat:
·
Select alactating rat, in the third to twenty-first day after
parturition and weighing about 300g separate it from the litter and 30 to 60
minutes later anaesthetise (for example, by the intraperitoneal injection of a
solution of Pentobarbitone Sodium).
·
Tie the rat to anoperating table, maintained at 37, by its hind legs
leaving the front legs free. Cannulate the trachea with a short polyethylene
tube of internal diameter about 2.5 mm in such a manner so as to ensure a free
airway; apply artificial respiration only if necessary.
·
Cannulate an external jugular or femoral vein with a polyethylene tube
of internal diameter about 0.4 mm which is filled with saline solution and
closed with a pin.
·
Shave the skin surrounding the inguinal and abdominal teats and excise
the tip of one teat, preferably the lower inguinal teat. Insert a polyethylene
tube of internal diameter about 0.3 mm and external diameter about 0.6 mm, to a
depth sufficient to obtain appropriate measurement of pressure (3 to 10 mm
depth), into the primary teat duct which opens onto the cut surface and tie
firmly in place with a ligature.
·
Connect this cannula with a suitable strain gauge transducer (such as
that used for recording arterial blood pressure in the rat) and fill the whole
system with a 3.8% w/v
·
solution of sodium citrate or saline solution containing 50 Units of
heparin sodium per ml to prevent clotting of milk.
·
After cannulation, inject a small volume (0.05 to 0.2 ml) of this
solution into the teat duct through the transducer to clear the milk from the
tip of the cannula. (This procedure may be repeated during the assay should
obstruction arise rom milk ejected into the cannula).
·
Clamp the strain gauge so that a slight
tension is applied to the teat and its natural alignment is preserved and
connect the gauge to a potentiometric recorder adjusted to give full-scale
deflection for an increase in milk-ejection pressure of about 5.3kPa.
·
Inject all solutions through the venous
cannula using a 1 ml syringe graduated in0.01 ml and wash them in with 0.2 ml of
saline solution.
·
Prepare a solution of the Standard
Preparation and a solution of the preparation being examined in saline so that
the volume to be injected is between 0.1 ml and 0.4ml.
·
Choose two doses of the Standard
Preparation such that the increase in milk ejection pressure is about 1.35 kPa
for the lower dose and about 2.7 kPa for the higher dose.
·
As an initial approximation, a lower dose
of between 0.1 and 0.4 milli Unit and an upper dose of1.5 to 2 times this
amount may be tried.
·
Choose two doses of the preparation being
examined with the same inter-dose ratio, matching the effects of the doses of
the Standard Preparation as closely as possible.
·
Inject the four doses (two doses of three
Standard Preparation and two doses of the preparation being examined) at
intervals of 3to 5 minutes.
·
The two doses of Standard Preparation and
the two doses of the preparation being examined should be given according to a randomized
block or a Latin square design and at least four responses to each should be recorded.
·
Measure all the responses and calculate
the result of the assay by standard statistical methods.
BIOASSAY
OF VASOPRESSIN:
Principle:
The vasopressor activity is estimated by
comparing the activity of the preparation being examined with that of the
Standard Preparation of arginine vasopressin under the conditions of a suitable
method of assay.
Standard
Preparation:
·
The Standard Preparation
is the Ist International Standard for Arginine vasopressin,
established in 1978, consisting of freeze-dried synthetic arginine vasopressin
peptide acetate with human albumin and citric acid(supplied in ampoules
containing 8.20 Units),or another suitable preparation the potency of which has
been determined in relation to that of the International Standard.
·
Inject slowly into the
tail vein of a male albino rat weighing about300 g a solution of a suitable
alpha-adrenoceptor blocking agent. for example, 10 ml per kg of body weight of
a solution prepared by dissolving 5 mg of phenoxy benzamine hydrochloride in
0.1 ml of ethanol (95%), adding 0.05 ml of 1M hydrochloride acid and diluting
to 5 ml with saline solution.
·
After 18 hours,
anaesthetize the rat with an anaesthetic that will maintain a prolonged and
uniform blood pressure. After 45 to 60minutes, tie the rat on its back to the
operating table by its hind legs.
·
Cannulate the trachea
with a short polyethylene tube of external diameter about 2.5 mm and dissect a
carotid artery ready for cannulation. Then cannulate the femoral vein close to
the in Guinea ligament.
·
Retract the abdominal
muscles to expose the inguinal ligament. Retract the superficial pudendal vein
to one side & dissect the femoral vein towards the in guinea ligament from
the corresponding artery.
·
When dissecting, a deep
branch reaching the femoral vein must be found and tied off to prevent bleeding
during cannulation. Tie a short polyethylene cannula of external diameter about
1 mm into the femoral vein by two ligatures and join by a short piece of
flexible tubing to a 1-ml burette with an attached this funnel containing
saline solution at about 37.
·
Firmly fix a wet
absorbent cotton swab to the thigh so as to cover the incision and cannula.
·
At this stage inject
through the venous cannula 200 Units of heparin, dissolved in saline solution, per
100 g of bodyweight.
·
Then tie in a carotid
cannula of external diameter about 1 mm and connect by a column of saline
solution containing heparin with a suitable pressure measuring device such as a
mercury manometer of internal diameter about 2 to 3 mm.
·
The central and
peripheral nervous system including both vagus and associated sympathetic
nerves is left intact. No artificial respiration is necessary.
·
Taking care that no air
is injected, inject all solutions through the venous cannula by means of a 1-ml
syringe graduated in 0.01 ml and wash in with 0.2 ml of saline solution from
the burette.
·
Dilute the extract of the
Standard Preparation and the preparation being examined with saline solution so
that the volume to be injected is between 0.1 ml and 0.5 ml. Choose two doses
of the Standard Preparation such that the elevation of the blood pressure is
about 4 kPa for the lower dose and about 7 kPa but always submaximal for the
higher dose, the ratio of low to high dose being determined by the response and
usually being 3to 5.
·
As an initial
approximation doses of 3 and 5 milli Units may be tried. Choose two doses of
the preparation being examined with the same inter-dose ratio, matching the
effects of the dose of the Standard Preparation as closely as possible.
·
Inject doses at intervals
of 10 to 15 minutes. The two doses of the Standard Preparation and the two
doses of the preparation being examined should be given in a randomized block or a Latin square design and
four to five responses to each should be recorded.
·
Measure all the responses
and calculate the result of the assay by standard statistical methods.
BIOASSAY OF ACTH:
·
ACTH (Adrenocorticotropic hormone,
corticotropin) is polypeptide tropic hormone (39 amino acids) secreted by the
anterior pituitary gland.
·
ACTH stimulates the
production of cortisol, a steroid hormone important for regulating glucose,
protein and lipid metabolism, suppressing the immune system response, and
helping to maintain blood pressure.
Official Preparations
Corticotropin injection: Is a sterile solution , in a suitable diluent, of the polypeptide from the pituitary glands of · mammals. Potency range should be 80.0 – 120.0 % of USP corticotrophin units.
·
Corticotropin for
injection, antimicrobial agent. Repository corticotropin injection is
corticotropin in a sterile solution of partially hydrolyzed gelatin and is
intended for subcutaneous and intramuscular use. This solution has been adopted
as the reference standard for the bioassay.
o Packing: Preserve in single-dose or multiple-dose containers of Type-1glass.
o Storage: Store in cold place.
o Labeling: Injection recommends intravenous administration
Purpose
and rationale
·
This is a historical
assay method. Administration of pituitary ACTH decrease the ascorbic acid
present in the adrenals. The depletion of adrenal ascorbic acid is a function
of the dose of ACTH administered. This relationship has been used for a
quantitative assay of ACTH.
Solutions:
·
Solution A:
Five units of test or standard dissolved in 0.25 ml of 0.5% phenol solution and
diluted with 8.1 ml of 15% gelatin solution (Now 0.5ml contain 300 mU ACTH)
·
solution B:
Three ml of solution A diluted with 6 ml gelatin solution. Now concentration
reduced to 100 mU ACTH/ 0.5 ml.
·
solution C: Again
3 ml of solution B diluted with 6 ml of gelatin solution, the resulting
solution contains 33 mU ACTH/ 0.5 ml.
Procedure:
·
Male Wistar rat (100-200
g) are hypophysectomies (pituitary gland removed by surgery) one day prior to
the test.
·
For one test with 3 dose of test preparation
and standard solution used for the study.
·
Number of hypophysectomies
rats required: at least 36(preferably 60).
·
The hypophysectomies rats
are randomly distributed in to six groups. Each rat receives subcutaneous 0.5
ml of the various concentrations of test or standard.
·
Three hours after
injection, the animals are anesthetized and both adrenals removed, freed from
extraneous tissue and weighed. The rats are sacrificed and the scull opened
to verify completeness of hypophysectomy.
·
The adrenals are
homogenized in glass tubes contains 200 mg pure sand and 8.0 ml of 4%
trichloroacetic acid and the ascorbic acid determined. (Roe and Kuether 1943).
·
The potency ratio
including confidence limits is calculated with the 3 + 3 point assay.
Ascorbic acid determination:
Reagents
·
0.02% ascorbic acid solution
·
85 % sulfuric acid (9N H2SO4)
·
0.02 g/ml of dinitrophenolhydrazine in 9N H2SO4
·
0.06 g/ml of thiourea are dissolved in distilled water
·
Charcoal
Preparation of 0.02% ascorbic acid solution
·
100 mg L-ascorbic acid
are dissolved in 100 ml of 4% trichloroacetic acid (1mg/ml solution) (Solution
A= 1 % solution)
·
2 ml of Solution A
diluted in 10 ml of 4% trichloroacetic acid to achieve a 0.2% ascorbic acid
solution (solution B).
·
1 ml of solution B diluted in 10 ml of 4% trichloroacetic acid to
achieve a 0.02% ascorbic acid solution (solution C)
Preparation of other solutions
·
Sulfuric acid (85%) is obtained by adding 900 ml concentrated sulfuric
acid to 100 ml distilled water.
·
Two g dinitrophenylhydrazine are dissolved in 100 ml 9 N H2SO4 (75 ml
distilled water and 25 ml concentrated sulfuric acid).
·
6 g thiourea are dissolved in 100 ml distilled water. Calibration
·
Trichloroacetic acid (4%) is added to 0.0, 0.5, 1.0, 2.0, 3.0, 4.0, 6.0,
8.0 ml of the 0.02% ascorbic acid solution (solution C) and 1.0, 1,5 and 2.0 ml
of the 0.2% ascorbic
acid solution to reach a final volumeof 8.0 ml (Solution B).
·
100 mg charcoal is added to each sample and thoroughly mixed by shaking
for 1 min.
·
After 5 min the solutions are filtered.
·
An aliquot of 0.1 ml of the 6% thiourea solution is added to 2.0 ml of
the filtrate followed by 0.5 ml dinitrophenylhydrazine solution.
·
The mixture is shaken and heated for 45 min at 57°C in a water bath.
·
The solutions are placed in an ice-cold water bath and with further
cooling 2.5 ml of the 85% sulfuric acid are added. The curve is established at
a wave length of 540 mm using the solutions without ascorbic acid as blank.
BIOASSAY OF d-TUBOCURARINE:
1. Rabbit Head-drop Method :
Principle:
·
d-Tubocararine hydrochloride is injected into the marginal vein of a
rabbit’s ear till the rabbit’s neck muscles are relaxed such that the animal
cannot hold its head up.
·
The total amount of test sample required to produce the endpoint is
compared with the total amount of the standard sample required to produce similar
endpoint.
Selection of Rabbits:
·
Rabbits weighing 2 kg are used.
Animals should be free from disease, obtained from a healthy colony and should
be accustomed with the experimental procedure.
Experimental Procedure:
·
Rabbit is placed in a holder with its head
protruding outside. The head should be freely movable. Minimum 8 rabbits are
used. They are divided into two groups each containing 4 rabbits.
·
First group will receive standard sample
and the second group will receive the sample under test. d-Tubocurarine
solution is injected at a constant speed by infusion apparatus through the
marginal vein.
·
Injection should be given at a rate of
0.4ml/min and should take about 10 min.
Dose: 0.012% w/v in saline.
·
Infusion is continued till the rabbit will
not be in a position to hold its head erect or there will be no response by
focusing light on the eyes. Rabbits recover immediately from the effect of curarization.
·
During the experiment there is a
possibility of respiratory embarrassment which is treated by injecting
neostigmine methyl sulphate(0.05 mg.) and atropine sulphate immediately through
the marginal ear vein.
·
Cross-over test is carried out to minimize
biological error due to animal variation.
·
Those rabbits which received the standard
sample on the first day will be given test sample on the second day of
experiment and vice versa.
·
Mean dose which produces head drop of the
test sample is compared with the mean dose of standard preparation.
Frogs Rectus
Abdominis muscle Preparation:
·
A frog is pithed and laid on its back on a
cork covered board to which it is pinned.
·
The skin covering the abdomen is cut away
and the rectus abdominis muscle of one side is dissected from the pelvic girdle
to its insertion in the cartilage of the pectoral girdle.
·
The muscle is then pinned to the cork by four
pins to keep its normal length while a thread is sewn through each end.
·
It is then mounted in the organ bath containing
frog's Ringer solution which contains:
NaCl, 6.5gm.; KCl, 0.29 gm.; CaCl2, 0.24 gm.; NaHCO3, 0.4 gm.; glucose, 1.5 gm.
and distilled water 2000 ml.
·
Oxygenation is carried out to keep the
tissue alive. The muscle is stabilized for 30-45 min. in order to get critical
quantitative response.
·
The responses are recorded using isotonic
frontal writing lever with 1 G. tension.
·
Two similar contractions with the same
concentration of acetylcholine are obtained.
·
Three doses of the standard sample and one
intermediate dose of the test sample are selected and the reduction in height
of contraction induced by acetylcholine is noted down.
·
Acetylcholine contraction is recorded on
slow moving drum for 90 second. d-Tubocurarine is allowed to act for 30 sec.
·
The percentage reduction at each dose
levels is calculated and log dose response curve of the standard drug is
plotted. A linear response will be obtained. The potency of test sample is
calculated from the standard curve.
BIOASSAY OF
DIGITALIS:
Principle:
·
Potency of the test sample is compared
with that of the standard preparation by determining the action on the cardiac
muscle.
Standard Preparation and Units:
·
The standard preparation is a mixture of
dried and powdered digitalis leaves (1 unit =76 mg.)
Preparation of Extracts:
·
Exact amount of the powder is extracted
with dehydrated alcohol in a continuous extraction apparatus for six hours. The
final extract should contain 10 ml(5 ml. alcohol +5 ml. water) per 10 g. of
digitalis powder. It should be stored in between 5oC and – 5oC.
1. Guinea-pig Method (Endpoint method):
·
Standard and test sample extracts are diluted
with normal saline in such a way that 1g of digitalis powder is diluted to 80
ml.
·
A guinea pig is anaesthetized with a
suitable anesthetic. It is dissected on the operation table. The jugular vein
is traced out by removing adhering tissues and cannulated by means of venous
cannula. A pin is inserted in the heart, such that it gets inserted in the apex
of the heart. In this way, we can observe the heart beats by up and down
movements of the pin.
·
The injection is continued through venous
cannula until the heart is arrested in systole. The amount of extract required
to produce this effect is taken as the lethal dose of the extract.
·
Another set of 19 animals of the same
species are used for this experiment and the average lethal dose is determined.
It is not necessary to determine the lethal dose of the standard during each
time of the experiment. But it should be occasionally checked.
·
The
lethal dose of the test sample is determined in a similar way using minimum
6guinea-pigs of the same strain.
·
The potency of the test sample is
calculated in relation to that of the std. preparation by dividing the average
lethal dose of the sample to the test and expressed as units per gram
BIOASSAY OF
HISTAMINE:
·
Histamine is present in almost all the
animal tissues mostly within mast cell and basophil granules. Tissues rich in histamine
are skin, gastric and intestinal mucosa, lungs, liver and placenta.
·
Non-mast cell histamine is present in
brain, epidermis, gastric mucosa and growing regions. Histamine is also present
in blood, most body secretions, venoms and
pathological fluids. It is now known to play important physiological roles.
·
Histamine produces effects by acting on
the histamine receptors (H1, H2, H3 and H4) present on target cells
·
Bioassay of histamine on isolated guinea
pig ileum can be determined by graded bioassay procedure i.e.
i)
Matching bioassay,
ii)
Interpolation bioassay,
iii)
Bracketing assay,
iv)
Multiple point assays.
·
Bioassay of histamine in biological
samples can be studied by using different bioassay methods. Depending upon
pharmacological action, histamine can be assayed by:
o Contractile effect of isolated ileum of guinea pig,
o Contractile effect of uterus of guinea pig and
o Fall in blood pressure of anaesthetized and atropinized cat.
Guinea pig's
uterus Bioassay of histamine using guinea pig ileum:
Principle:
·
Guinea pig ileum is very useful
preparation for the bioassay methods. It is more sensitive to histamine.
Contractile response of histamine to ileum is due to presence of H1 receptor.
Preparation of Standard and other solution:
·
Prepare the stock of Tyrode solution. Also
prepare the standard stock solution of histamine (1 mg/ml) and then different
concentrations of histamine by serial dilution method.
Procedure:
·
Sacrifice the 24 hr fasted guinea pig by
stunning on the head and carotid bleeding. Fix the animal on the dissecting
board by tying its legs. Open the abdominal cavity by a small horizontal cut
followed by vertical midline incision and expose the abdominal organs.
·
Trace the ileocecal junction by lifting
the caecum, then go upwards up to 8 cm from the junction and cut the 2-3 cm
long segment of ileum muscle (excluding the terminal 5-8 cm, contains excess of
excitatory α-adrenergic receptor, hich may interfere in the study).
·
Immediately place it in a petri dish
containing aerated warm Tyrode solution. Slowly remove the mesentery attached
to the muscle and then gently clean the lumen of ileum by passing luke warm
Tyrode solution through it with a pipette or syringe.
·
Mount the preparation in the inner organ
bath containing Tyrode solution (20 ml) maintained at 37ºC. Tie the bottom end
of the muscle to the hook of aeration tube and the upper end to the isotonic
frontal lever by a thread without closing the lumen
·
Adjust the magnification of response to 7-10-fold. Aerate the tissue
with O2 or carbogen slowly (40-60 bubbles per minutes).
·
Use 5 min time cycle with contact time of 30s for recording the
contraction dependent responses of tissue due to histamine on the smoked drums.
·
Record the responses of standard and test compounds.
·
Record the responses of test compound i.e. unknown concentration of
histamine with gradually increasing volume, till obtaining a response (T) which
would lie on the linear portion (30-70%) of the CRC. Fix the response obtained
due to volume of T.
·
Record the graded responses of standard solution and test solution of
histamine.
·
The potency of the test sample is calculated in relation to that of the
std. preparation by dividing the average lethal dose of the sample to the test
and expressed as units per gram.
BIOASSAY OF 5-HT:
·
5-Hydroxytryptamine (5HT) is the biologically active local hormone, low
molecular weight, and also an important neurotransmitter in the brain and
periphery.
·
It was first detected in serum (serotonin) and enterochromaffin cells of
gut mucosa (enteramine), and later both were identified to be 5HT. Today the
terms 5HT and serotonin are used interchangeably.
·
About 90% of body's content of 5-HT is localized in the intestines; most
of the rest is in platelets and brain. It is also found in wasp and scorpion
sting, and is widely distributed in invertebrates and plants (banana, pear,
pineapple, tomato etc.).
·
The concentration of 5-HT in biological samples can be assayed by:
Isolated fundus strip of rat stomach, Isolated terminal colon of rat, Isolated
rat uterus, Perfused rabbit ear.
Isolated fundus strip of rat stomach
Principle:
·
Rat fundus preparation is a very sensitive tissue for the bioassay
methods and is useful to study the action of several substances like 5-HT, ACh,
PGE2 and bradykinin.
·
Fundus strip preparation is slow contracting muscle.
·
Fundus part of stomach can be easily identified by its grey colour and
situated above the pink coloured thick pyloric part. A zig-zag preparation of the
fundal strip is prepared so as to expose maximum portion of the tissue to drug.
Preparation of
Standard and other solution:
·
Prepare the stock of Krebs solution.
Prepare the standard stock solution of serotonin (1 mg/ml) and then different
concentrations of serotonin by serial dilution method.
Procedure:
·
Sacrifice the 24 hr fasted rat by stunning
on the head and carotid bleeding. Fix the animal on the dissecting board by
tying its legs. Open the abdominal cavity by a small horizontal cut followed by
vertical midline incision and expose the abdominal organs.
·
Identify the stomach and separate it from
the abdomen by gently cutting its cardiac and pyloric end. Place it in a petri
dish containing aerated warm Krebs solution.
·
Incise the fundus of the stomach (upper
grey part) from pyloric part (pink and thick part). Cut the fundus from the
lesser curvature and open it longitudinally. Then cut the fundus at the midline
into two equal parts. Give alternate transverse cuts (zig-zag cut) on opposite
sides of the muscle to make a fundal strip preparation.
·
Mount the preparation in the inner organ
bath containing Krebs solution (20 ml) maintained at 37ºC. Tie the bottom end
of the muscle to the hook of aeration tube and the upper end to the isotonic
frontal lever by thread. Adjust the magnification of response to 10-15 fold.
Aerate the tissue with O2 or carbogen slowly (40-60 bubbles per minutes).
·
Apply 1 g load and stabilize the tissue
for 30 min during which wash the tissue with fresh Krebs solution once in every
10 min.
·
Use a 5 min time cycle with contact time
of 90s (since the fundus strip muscle contracts slowly and relaxes slowly) for
recording the contraction due to serotonin.
·
Record the graded responses of standard
solution and test solution of serotonin.
·
The potency of the test sample is
calculated in relation to that of the std. preparation by dividing the average
lethal dose of the sample to the test and expressed as units per gram.
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