Bioassay of d-tubocurarine phrenic nerve method and Head drop method

1. BIOASSAY
OF
d- TUBOCURARINE
KOPPALA RVS CHAITANYA
ASSISTANT PROFESSOR
DEPARTMENT OF PHARMACOLOY

2. The activity of a sample of d - tubocurarine chloride is determined by comparing its activity with
that of std preparation of d-tubocurarine chloride by a biological method. The sample passes the test if
its potency does not differ from that of std preparation by more than 10%.
Standard preparation: The standard preparation is a quantity of pure crystalline d-tubocurarine
chloride.
Suggested method:
Phrenic nerve –diaphragm method:
➢ The phrenic nerve of a rat is dissected out on one side and cut off high up in the thorax and the
corresponding anterior quadrant of the diaphragm is cut out and attached by its coastal margin to a
bent glass rod immersed in a bath containing a solution of following composition:
1. sodium chloride -9.0gm
2. potassium chloride -0.42gm
3. calcium chloride – 0.24 gm
4. sodium bicarbonate – 0.5gm
5. dextrose-1.0 gm
6. water (redistilled and condensed in glass) -1000 ml
➢ The bath is maintained at a temperature of 37°C and suitably oxygenated. The central tendon is tied
to a lever recording on a smoke drum. The name is electrically stimulated through suitable electrodes
with the maximal shocks of less than one milli second duration, at intervals of 10 to 15 seconds.
➢ Suitable dose of d-tubocurarine chloride, usually from 0.125mg to 0.175mg for a bath volume of
100ml, when added to the solution in bath, produces a reduction in the height of contraction as
recorded on the drum. doses of standard preparation are added at the ten-minute intervals, and are
allowed to act for exactly 5 minutes. The preparation is washed twice between doses with warm fresh
solution of the above composition unit the height of contraction becomes constant for the same dose.
➢ A high and low dose of the standard preparations are chosen so that the % reduction 75 and 25
respectively. A similar dose preparation for test are chosen. The 4 doses, 2 of the standard and 2 tests
are then administered at 10-minute interval in an order represent by the rows of 4*4 Latin square,
the doses are assigned at random. Each dose administered 4 times.

3. ➢ The average effect is determined and the potency is estimated by the standard method for a 2*2
assay, log of doses being used.
Head drop method:
➢ The solution of d-tubocurarine chloride to be administered (which to be tested) into the ear vein of
rabbit until no longer hold up its head. The amount drugs.
➢ The amount of drug required to produce this end point is noted. The assay is carried out as a “cross
over” test on several rabbits, the unknown and standard preparation being administered to each
animal on different days( in case of respiratory embarrassment is severe, an injection through the
original puncture in the ear vein of neostigmine methylsulphate (0.5mg) and atropine sulphate
(0.5mg) will rapidly restore normal respiration.
➢ Since rabbits vary significantly in their sensitivity to tubocurarine, the assay is done as a “cross over
test”.
➢ The required limits of error may be expected to be obtained using a total of 8 animals for the test.
➢ Limits of errors: 85% and 115% ( p= 0.95)