Brit. J. Pharmacol. (1955), 10, 208.
THE MODE OF ACTION OF SULPHONAMIDES, PROGUANIL
AND PYRIMETHAMINE ON PLASMODIUM GALLINACEUM
I. M. ROLLO
From the Wellcome Laboratories of Tropical Medicine, 183, Euston Road, London, N.W.J
(RECEIVED JANUARY 3, 1955)
Despite the mass of information on the anti-
malarial action of sulphonamides, proguanil, and
pyrimethamine (see Goodwin and Rollo, 1955),
there is as yet no complete picture of the relation-
ship between them, although they all probably act
upon the same metabolic pathway in the synthesis
Hawking (1953a) in his review
of protozoal chemotherapy pointed out: " Many
different lines of work appear to be converging
here towards a general explanation, but it will be
necessary to achieve further elucidation of the
metabolism of p-aminobenzoic acid (PAB) and of
folic acid by the malarial parasite before all the
different facts in the jigsaw puzzle can be fitted
In this paper an attempt is made to
fill some of the gaps in our knowledge of cross-
resistance, potentiation, and antagonism between
antimalarial drugs, by reviewing and analysing the
known facts, in the light of new data.
The parent strain of Plasmodium gallinaceum was
that maintained in these laboratories for many years
by blood and occasional mosquito passage in young
This and the other drug-treated strains have,
during the course of the experiments, been passaged
solely by blood inoculation.
chicks (Rhode Island Red-Light Sussex cross) were in-
oculated intravenously with approximately 50 million
parasitized red blood cells.
were given orally either in solution or, if insoluble in
water, in gum tragacanth suspension.
hours after inoculation, a total of seven doses was
given over 3-} days.
Infection was assessed from
stained blood films on the fourth day after inoculation,
when in untreated controls about 70-90% of the red
blood cells were infected.
the test animals were counted and the results were ex-
pressed as percentages of the controls.
which reduces parasitaemia to 50% of the mean para-
sitaemia of untreated controls (ED5O) was obtained
from a 3- or 4-dose assay (Rollo, 1952).
five chicks was normally used at each dose level.
Cross-resistance.-A strain of P. galinaceum (P.36),
Five- or twelve-day-old
The antimalarial drugs
Starting a few
The infected red cells in
A group of
pyrimethamine, was obtained from Dr. D. G. Davey,
of Imperial Chemical Industries.
this strain to sulphadiazine was tested in order to
complete the picture of cross-resistance relationships
reviewed by Thurston (1953).
were prepared by treating successive passages with sub-
curative doses of proguanil and pyrimethamine respec-
During each passage the chicks received a total
of seven doses of the drug as described above. Both
strains were passaged and treated in parallel and were
tested periodically for cross-resistance during the early
stages of the development of drug resistance.
Potentiation.-The potentiating effect of pyrimeth-
amine upon the activity of proguanil was investigated
by giving the drugs both singly, and together in
to groups of infected chicks.
ED50's were determined from the dose-response curves
and were plotted on a graph to demonstrate the effect
of one drug upon the action of the other.
periments were done using pyrimethamine with sulph-
adiazine, sulphaguanidine, succinylsulphathiazole, peni-
cillin, or streptomycin. In some experiments the blood
levels of the sulphonamides produced by single oral
doses were determined by the method of Bratton and
Inhibition.-The effects of PAB and folic acid on
the activity of sulphadiazine and pyrimethamine were
The PAB or folic acid was given intra-
peritoneally in aqueous solution or suspension 30 min.
before each oral dose of the antimalarial drug except
in one experiment with pyrimethamine in which PAB
was given orally five times a day at three-hourly inter-
vals; in addition, the chicks were fed on a diet con-
taining 0.1 % PAB.
To ensure that the diet would be
consumed at night when the chicks were not being
dosed, the cages were darkened throughout the day and
brightly lit during the night.
The inhibitory action of amino-an-fol (2, 4-diamino-
pteroylaspartic acid) was investigated in one experi-
The sensitivity of
Two further strains
Cross-resistance.-The effect of sulphadiazine
upon strain P.63 is shown in Table I. There was
no evidence of resistance to sulphadiazine although
this strain was highly resistant both to proguanil
1. M. ROLLO
action involving, firstly, an acceptor mechanism
whereby the drugs are made available to interfere
with the metabolic
" lethal " point of action within the metabolic path-
way-probably by interfering with the conversion
of folic to folinic acid.
The author is indebted to Miss G. Horton, Miss J. D.
Hughes, and Miss C. Reynolds for their assistance in
the laboratory; to Dr. G. H. Hitchings for his criticism
and helpful advice;
to Dr. L. G. Goodwin for his
encouragement and help in preparing the manuscript;
and to the Wellcome Foundation for permission to
publish the experimental data.
The amino-an-fol used was the generous gift ofDr. W.
Jacobson, Strangeways' Research Laboratories, Cam-
bridge, and the synthetic folic acid was kindly provided
by Dr. T. Jukes, Lederle Laboratories.
Brit. med. Bull., 8, 47.
Bishop, A. (1951).
and McConnachie, E. W. (1948).
Bratton, A. C., and Marshall, E. K. (1939).
Chem., 128, 537.
Broquist, H. P., Kohler, A. R., Hutchison, D. J., and
Burchenal, J. H. (1953).
Coatney, G. R., Cooper, W. C., Eddy, N. B., and
Greenberg, J. (1953). Survey ofAntimalarial Agents,
p. 252, U.S. Public Health Monograph No. 9.
Parasitology, 40, 175.
Ibid., 40, 163.
Ibid., 202, 59.
Emerson, S., and Cushing, J. E. (1946).
Goodwin, L. G., and Rollo, I. M. (1955). In Biochemistry
andPhysiology ofProtozoa, vol. 2, ed. Hutner, S. H.,
and Lwoff, A.
New York: Academic Press.
Greenberg, J. (1949).
J. Pharmacol., 97, 238.
Exp. Parasit., 2, 271.
Ibid., 3, 351.
and Bond, H. W. (1954).
Hawking, F. (1953a).
Symposium " Growth Inhibition
and Chemotherapy," VIth International Congress
of Microbiology, Rome.
Brit. med. J., 1, 1201.
Landy, M., Larkum, N. W., Oswald, E. J., and Streight-
off, F. (1943).
Science, 97, 265.
Maegraith, B. G. (1953).
Brit. med. J., 2, 1047.
Trans. R. Soc. trop. Med. Hyg., 48, 275.
Maier, J., and Riley, E. (1942).
N.Y., 50, 152.
Refaat, M. A., and Bray, R. S. (1953).
Robertson, G. I., Davey, D. G., and Fairley, N. H.
Ibid., 2, 1255.
Rollo, I. M. (1951).
Nature, Lond., 168, 332.
Trans. R. Soc. trop. Med. Hyg., 46, 474.
Singh, J., Ray, A. P., Basu, P. C., and Nair, C. P. (1952).
Ibid., 46, 639.
Thurston, J. P. (1953).
Parasitology, 43, 246.
Ibid., 44, 99.
Work, T. S., and Work, E. (1948).
therapy, p. 268. London: Oliver & Boyd.
Fed. Proc., 5,
J. Parasitol., 40, 1.
Proc..Soc. exp. Biol.,
Brit. med. J..
The Basis ofChemo-