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
MODE OF ACTION OF ANTIMALARIAL DRUGS
evidence of hypersensitivity to sulphadiazine.
The pattern of development of resistance in the
two other strains which had been treated in parallel
THE ACTION OF SULPHADLAZINE ON NORMAL AND
PROGUANIL-PYRIMETHAMINE RESISTANT STRAINS OF
*Parasitized cells in the treated birds shown as a percentage of
the parasitized cells in the untreated controls.
percentage the more effective is the particular dose.
Hence the lower the
CROSS-RESISTANCE TESTS BETWEEN PROGUANIL- AND
PYRIMETHAMINE-TREATED STRAINS OF P. GALLINACEUM
*As in TableI.
The proguanil-treated strain soon acquired high
resistance, and at the same time slight but definite
cross-resistance, to pyrimethamine.
the strain treated under the same conditions with
pyrimethamine had not become distinguishable
from the parent strain in sensitivity to either drug.
Potentiation.-Table III shows the
different proportions to groups of infected chicks.
The ED5O's are plotted in Fig. 1.
below the straight line joining the ED5O's of the
At this time,
THE COMBINED ACTION OF PROGUANIL AND PYRI-
METHAMINE ON P. GALLINACEUM
t EDS0 proguanil alone
percentage parasitaemia as in Table I.
Uj ~ ~~~~
ED50 PYRIMETHAMINE (pg./kg.)
FIG. 1.-ED50's (doses reducing parasitaemia to 50% of the mean
parasitaemia of untreated controls) of proguail and pyrimeth-
amine administered both singly and together in various propor-
tions, in chicks infected with P. gallinaceum.
graphically from dose-response curves.
Note lack of potentiation.
For interpretation see
PYRIMETHAMINE ON P. GALLINACEUM
7__ - S
*Untreated controls; percentage parasitaemia as in Table I.
t EDS0 sulphadiazinealone 26mg./kg.
1. M. ROLLO
FDG. 2.-ED50's of pyrimethamine and sulphadiazine, administered
and together in various proportions, in chicks infected with P. M
Conventions as in Fig. 1. Note marked potentiation.
two drugs given alone indicate potentiation.
action of pyrimethamine was not potentiated by
proguanil (Fig. 1), but was greatly potentiated by
sulphadiazine (Table IV, Fig. 2). The point on the
curve nearest the origin indicates the optimum
combination of doses which
Here this proportion is 1/7th of
the ED50 of sulphadiazine with 1/8th of the ED50
The potentiating action of sulphadiazine might
possibly be due to an action upon intestinal organ-
isms which normally synthesize metabolites essen-
tial to the malarial parasite.
poorly absorbed sulphonamides and antibiotics
which act upon the intestinal flora is shown in
will produce the
The effect of giving
THE EFFECT OF POORLY ABSORBED SULPHONAMIDES
AND ANTIBIOTICS ON THE ANTIMALARIAL ACTION OF
PYRIMETHAMINE ON P. GALLINACEUM
that the potentiating effect was due,
not to the effect in the gut, but to the
sulphonamide in the blood.
sulphathiazole was found in the blood
only at a very low level and did not
reached a much higher level and did
potentiate.The antibiotics had no
Inhibition.-The inhibitory action of
PAB and folic acid upon the activity
of sulphadiazine and pyrimethamine is
shown in Table VI.
was essentially competitive in nature.
The effect of PAB upon pyrimeth-
amine was barely significant (P=0.1)
because of the large variance of the
results.Repetition of the experiment
gave similar figures.
antagonism was small, perhaps because
pyrimethamine persists in the blood
and is therefore difficult to antagonize
with a compound which is probably rapidly elimi-
Further work is necessary to investigate
this aspect of the problem.
Results obtained using amino-an-fol with sulpha-
diazine are also included in Table VI; it acted in
the same manner as folic acid. Although chromato-
graphic examination of the sample did not dis-
close any PAB, there were impurities present and
these may have affected the result.
These results show clearly
The degree of
COMPETITIVE ANTAGONISM OF SULPHADIAZINE BY
PAB AND OF SULPHADIAZINE AND PYRIMETHAMINE
BY PGA IN P. GALLINACEUM
120 None| 0-03 l0-06
*Untreated controls; percentage parasitaemia as in Table I.
(Each rectangle enclosed in bold rules contains the results of a
separate experiment, i.e., this table is made upfrom 5experiments.)
*Untreated controls; percentage parasitaemiaas in Table I.
tA single oral dose of sulphadiazine at 10 mg./kg. gave a peak
blood level of 0.84 mg.%at I hr.
MODE OF ACTION OF ANTIMALARIAL DRUGS
Mode of Action of Sulphonamides.-It is com-
monly recognized from studies on bacteria that
sulphonamides containing an NH2.C6H4.SO2.NH-
group act by competing with PAB.
malarial action of sulphonamides may also depend
on a similar mechanism because PAB reverses it
(Maier and Riley, 1942) and because PAB is im-
portant for the metabolism of malarial parasites,
particularly P. berghei, which is extremely sensitive
to sulphadiazine (Hawking, 1953b).
Resistance and Cross-resistance.-There are at
least four possible explanations of the mechanism
organism may produce sufficient PAB to carry on
the inhibited metabolic reaction.
Larkum, Oswald, and Streightoff (1943) found that
resistant staphylococci produce up to 100 times
more PAB than did normal strains.
hibited reaction may be by-passed by a related
action not affected by sulphonamides; or may be
made unnecessary by the direct utilization of the
end-product of the reaction (Work and Work,
1948). (3)The resistant strain may develop enzymes
for the conversion
of sulphanilamide into an
essential metabolite, as suggested by the results
of Emerson and Cushing (1946) with sulphon-
of the cell membrane may be altered so that the
sulphonamides are selectively excluded; or spatial
rearrangement of cell receptors may restrict the
attachment of the sulphonamide molecule but not
that of PAB.
Thurston (1953) put forward the tentative sug-
gestion that sulphadiazine resistance in P. berghei
may be due to the increased production of PAB.
work, showing that malarial infections-particu-
larly with P. berghei-(Hawking, 1953b), do not
progress normally in the absence of dietary PAB,
synthesize PAB. The experimental conditions are
critical and any departure from a strict PAB-
deficient diet may give conflicting results (Mae-
graith, 1954); other substances besides PAB may
be involved in the suppression of malaria by milk
(Maegraith, 1953; Refaat and Bray, 1953).
upon several species of Plasmodium (Thurston,
1953), together with the results shown in Table I,
lead to the conclusions that
usually resistant to proguanil and to pyrimeth-
(1) The resistant
(2) The in-
(4) The permeability
(b) Strains resistant to proguanil are usually
resistant to pyrimethamine and vice versa.
Although some reported results conflict with
these conclusions there is usually a reason for the
Thus Thurston (1953) and Rollo
(1951) reported strains of P. cynomolgi and P.
gallinaceum respectively which, while resistant to
proguanil, retained sensitivity to pyrimethamine.
On the other hand, Robertson, Davey and Fairley
(1952) and Singh, Ray, Basu and Nair (1952) re-
ported strains of P. gallinaceum and P. knowlesi
which became cross-resistant to pyrimethamine
after treatment with proguanil.
by Thurston and Rollo were " old " strains which
retained a high degree of resistance to proguanil,
but which had not been exposed to that drug for
some time before
testing with pyrimethamine.
The strains used by Robertson et al. and Singh et
shortly before the cross-resistance tests were car-
Proguanil may, therefore, under optimal
conditions, give rise to resistance to pyrimethamine
as well as to proguanil.
appears to develop less readily, and is more labile
in character, than resistance to proguanil (Table
II); if the strain is left without treatment, sensi-
tivity to pyrimethamine may return.
Rollo (1951) showed that a strain of P. berghei,
treated in 9 successive passages with sulphadiazine
and rendered 4 times less sensitive to the drug
A similar strain, prepared over
a longer period by Thurston (1953), and showing a
100-fold increase in resistance to sulphadiazine,
strain, however, was only partially resistant, and
it is possible that, with an increase in its resistance
to sulphadiazine, cross-resistance to pyrimethamine
would have appeared.
Bishop and McConnachie (1948) reported that
a proguanil-fast strain of P. gallinaceum became
resistant to sulphadiazine.
found that strains made resistant to proguanil have
Bishop (1951) was unable to confirm the earlier
result. This cross-resistance is not dependent upon
the pyrimidine part of the molecule, for Bishop
and McConnachie (1950a) showed that a strain of
P. gallinaceum resistant to sulphanilamide was
also highly resistant to proguanil.
(1949) showed that the action of proguanil
The strains used
All other workers have
to sulphadiazine, and
I. M. ROLLO
strongly potentiated by sulphadiazine and other
PAB competitors which are themselves
against malaria. From the present work it is clear
that a similar relationship exists between pyrimeth-
amine and sulphadiazine, but that pyrimethamine
and proguanil do not potentiate each
It is likely that potentiation occurs when two
drugs act at different points on the same meta-
bolic pathway, although it is possible that indirect
effects-such as the reduction of available meta-
bolite by the action of drugs on the intestinal flora
-might produce the same result.
that an additive effect occurs when the drugs act
at the same point or upon different pathways.
different point from, but on the same pathway
as, pyrimethamine or proguanil.
relationship with sulphadiazine, and because their
antimalarial action is antagonized by PAB, it is
probable that proguanil and pyrimethamine act
at the same point on the same pathway.
The antagonism ofsulphadiazine by both PAB
and folic acid is competitive and can easily be
demonstrated. This has been shown in mice in-
fected with P. berghei by Thurston (1954) and in
infections of P. gallinaceum (see above). Antagon-
ism of proguanil or of pyrimethamine by PAB
and folic acid is less effective and less easy to
Greenberg (1953), using high doses
of PAB and folic acid, has shown in P. gallinaceum
that proguanil can be competitively antagonized.
Thurston (1954), by using a continuous dosing
technique, has shown that both proguanil and
pyrimethamine can be antagonized by PAB and
Thurston's data are too sparse to decide whether
the antagonism is competitive or not, except with
pyrimethamine and PAB, where competition
Both PAB and folic acid antagonize-
the latter competitively-the action of pyrimeth-
amine on P. gallinaceum (see above).
Thus in every example in which adequate data
have been collected, the compounds have acted
as competitive antagonists to all three drugs. The
sulphadiazine suggests that folic acid is broken
down either by the host or the malarial parasite
itself to PAB or p-aminobenzoylglutamic
The folic acid antagonists amino-an-fol, aminop-
terin, and amethopterin, are without antimalarial
activity (Coatney, Cooper, Eddy and Greenberg,
1953); the first of them has been shown above to
antagonize the action of sulphadiazine.
Greenberg (1954) has shown that folic acid, amino-
It is also likely
Because of their
an-fol, and several other folic acid antagonists,
competitively antagonize the action of sulpha-
diazine on P. gallinaceum.
ably unable to utilize preformed folic or folinic
acid and it is likely that they synthesize these from
PAB (Goodwin and Rollo, 1955), which may be
supplied by the breakdown of folic acid or of the
folic acid antagonists.
siders that it is uncertain whether the antagonism
of sulphadiazine by substances containing the
PAB moiety occurs because the analogues act as
sources of folic acid or because the analogues are
broken down to PAB which inhibits the sulphon-
Possible Mode of Action of Sulphonamides,
Proguanil and Pyrimethamine.-The
systems upon which we must assume the action
of these antimalarials to take place are shown
Plasmodia are prob-
Greenberg (1954) con-
Purine and pyrimidine
Purines and pyrimidines,
The facts to be discussed are: (1) strains resis-
tant to proguanil and pyrimethamine retain full
sensitivity to sulphonamides,
resistant strains are cross-resistant to proguanil
and pyrimethamine, and (3) sulphadiazine poten-
tiates the action of proguanil and pyrimethamine.
Although the first and third of these facts suggest
that'the loci of action of sulphonamides and pro-
guanil-pyrimethamine are not the same, the second
suggests a common locus.
The effect of sulphonamides on plasmodia is
presumably due to interference with the utilization
of PAB in reaction A.
however, act quite differently.
cross-resistance tests and the lack of complete
antagonism by PAB and folic acid indicate that
their action may be more than simple competition
with a metabolite.Hitching's evidence (in Good-
win and Rollo, 1954) on the varying ability of
folic and folinic acids to reverse the effects of
pyrimethamine favours a blockage of reaction B;
and if resistance to proguanil and pyrimethamine
involved interference with their action at point B
only, this would leave reactions A and C vulner-
able to other drugs-for example, to the action of
sulphonamides on reaction A.
are known that interfere with reaction C in plas-
Resistance to sulphonamides could be
attributed to a by-pass of the whole series of
reactions from PAB to folinic acid, thereby render-
The other drugs may,
The results of
So far no drugs