Enhanced sensitivity of P. falciparum to sulphalene as a consequence of resistance to pyrimethamine

230 TRANSACTIONS OF THE ROYAL SOCIETY OF TROPKAL MEDICINE AND HYGIENE. Vol. 63. No. 2. 1969.

ENHANCED SENSITIVITY OF P. FAKIPARUM TO SULPHALENE CONSEQUENCE OF RESISTANCE TO PYRIMETHAMINE The Harry

AS A

DANIEL C. MARTIN AND JOHN D. ARNOLD S. Truman Laboratory, Kansas City General Hospital and The University Missouri School of Medicine, Kansas City, Missouri 64108

of

The antimalarial actions of PABA inhibitors have been repeatedly studied since the pioneering work of DIAZ DE LEON in 1932. Two characteristics of sulphonamide antimalarial activity have limited its usefulness. First, the sulphonamides and sulphones have little or no effect against .P. uivax. Second, the clinical effect of the PABA inhibitors against P. falczparum infections has been relatively slow, and cure rates have been variable from study to study. This is illustrated by the early findings of prontosil in P. fulciparum: HILL and GOODWIN (1937) reported no relapses, but MENK and MOHR (1939) reported a very poor result on parasitaemia. The development of P. falciparum strains resistant to chloroquine has developed fresh interest in the PABA inhibitors. The sulphonamides and sulphones have shown promise against multi-drug-resistant strains, which are usually resistant to chloroquine, pyrimethmine, atabrine (mepacrine) and other folic acid antagonists. Against multi-resistant strains the sulphonamides and sulphones were more effective than the early sulphonamide studies (COXON, 1945; COGGESHALL et al., 1941; FINDLAY et al., 1946) or some studies on sulphones (SAINI, 1947) would have indicated. It has been difficult to reconcile the early lack of enthusiasm for the sulphones and sulphonamides with the results of more recent work (BASU et al., 1962; DEGOWIN et al., 1966). A recent study (MARTIN and ARNOLD, 1968b) has provided some insight into this seeming inconsistency. This study was carried out with the moderately longacting sulphalene. When sulphalene was used to treat volunteers infected with multire’sistant P. fuZc+rum (Camp strain), 8 of 8 were cured by a single 1 *O gramme dose. The clinical response was unexpectedly rapid. In contrast, however, a P. fazciparum with “normal” drug response (UG I strain) was treated with the same drug and was much less responsive. A single dose of 2.5 grammes of sulphalene cured only 3 of 6 volunteers, and in all patients the clinical response was uniformly slow. One explanation for these findings is that the resistance to one or several drugs has changed the organism to such an extent that it now has an increased susceptibility to the sulphonamide. Drug resistance is often believed to be accompanied by several additional phenotypic changes, some of which are less advantageous to the parasite. It is not often possible to use this approach in chemotherapy. This paper will report on the marked increase in sensitivity to sulphalene, which

This study was supported by U.S. Army Contract No. DA-49-193-MD-2545 from the U.S. Army Research and Development Command, Office of the Surgeon General. This paper is contribution No. 438 from the Army Research Program on Malaria. We would like to thank Sheriff Arvid Owsley and his staff for their cooperation in these studies. We are especially grateful to the inmate volunteers from the Jackson County Jail whose cooperation made this study possible.

DANIEL C. MARTIN AND JOHN D. ARNOLD

231

can be induced by making a “normal” P. fulciparum strain resistant to pyrimethamine, and will provide evidence that the pyrimethamine resistance factor of the multi-resistant strains is the cause of the increased dependence on PABA utilization. Materials and methods Volunteers 31 men (22 Negro and 9 Caucasian) volunteered to participate in this study after extensive written and oral explanation of the risks. All had a negative history for prior malaria infections, and a careful medical history and physical examanation preceded admission to the malaria research unit. Routine medical laboratory work included: haematocrit, white blood count, WBC differential, platelet count, blood glucose, blood urea nitrogen, alkaline phosphatase, SGOT and urine analysis. This routine laboratory work was carried out on admission, before treatment (antimalarial), 3 and 7 days after treatment, and then once a month until the patient left the study. Additional admission studies included: prothrombin time, serological tests, blood typing and tuberculin skin test. All infections, hospital care and tests were carried out at the clincal annex of the Harry S. Truman Laboratory of Comparative Medicine, in the Jackson County Jail at Kansas City, Missouri. Parasite strains 11 volunteers were infected with the P. fulciparum strain UG I, obtained from a child in Kampala, Uganda. Its sensitivity to drugs has been extensively studied and reported (MARTIN and ARNOLD, 1967). It is quite sensitive to pyrimethamine, although the dose required increases in proportion to the size of the parasite population (MARTIN and ARNOLD, 1968a). 11 volunteers were also infected with P. fdciparum UG I strain after the strain had an induced resistance to pyrimethamine-UG I (py. resist.). The induction of pyrimethamine resistance was extremely simple in this strain, since exposure to one inadequate dose of pyrimethamine was frequently sufficient to induce resistance to the highest safe human dose. Although the induced resistance to pyrimethamine was quite marked, it was not very stable. Passage through 2 volunteers without exposure to the drug almost always resulted in a return of sensitivity to pryimethamine toward normal. All volunteers infected with UG I (pyrimethamine-resistant) strain were infected from the same blood and infection source, and the resistance to pyrimethamine was confirmed by treating one patient with pyrimethamine 100 mg. There was no discernable, antimalarial effect from this dose. The 9 volunteers infected with P. fulciparum Camp strain and treated with sulphalene have been included for comparison. Procedures P The 31 volunteers were infected by intravenous injection of lo6 to lo7 parasitized red blood cells (RBC), either from freshly obtained blood or from a blood-citrateglycerin preparation which had been frozen in liquid nitrogen. Infected volunteers were observed in a hospital area and daily parasite counts were made before and during the initial parasitaemia. Thick smears were examined by the method of EARLE and PEREZ(1932), 3 times a week during 60 days of observation of all volunteers after treatment. Treatment with sulphalene was started after one day of fever and after 2 or more days of confirmed parasitaemia. 20 of the 31 volunteers had maximum parasitaemia in excess of 1,000 per c.mm. Among volunteers infected with P. fuhparum ‘(UG I, py. resist.) the maximum parasitaemias ranged from 130 to 13,830 parasites per cmm. of blood. Response to drug The location and conditions of this study preclude the possibility of malarial infections other than those which are experimentally induced. Infection by blood injection is presumed to preclude the possibility of exo-erythrocytic infection. Any

232

ENHANCED

SENSITIVITY

TO SULPHALENE

P.falciparum

IN PYRIMETHA~UNE-RESISTANT

reappearance of asexual parasites in the peripheral blood after treatment must then be due to the incomplete blood-schizontocidal effect of the drug. Recrudescences following the use of these drugs individually and in combination, have always occurred in less than 48 days. Results

The change in susceptibility to the sulphalene is quite striking; the “normal” strain of P. falczparum is relatively resistant, but induction of pyrimethamine resistance makes the strain significantly more susceptible. When 1.0 gramme of sulphalene was given as a single dose to volunteers infected with the normal strain, 3 of the 5 had a recrudescence. The speed of action for this drug was unusually slow compared with chloroquine; lysis of fever averaged 4 days and clearance of parasitaemia 8.4 days. Increasing the amount of sulphalene from 1.0 to 2.5 grammes (single doses) did not improve the performance; 3 of 6 volunteers had recrudescences after the increased dose. The 2.5 gramme dose did increase the rate of clearance of symptoms (3 days) and parasitaemia (6 days), but this is still quite slow. The performance of sulphalene against the UG I (pyrimethamine resistant) strain of P. fuhpurum was remarkably better than against the “normal” UG I strain. Sulphalene as a single 1.0 gramme dose cured 9 of 11 volunteers ; the fever was lysed in an average of 1.4 days, the parasitaemia cleared in an average of 3 days. This is an excellent and rapid response which is equivalent to the use of chloroquine against non-resistant strains. The data from these experiments, along with a parallel study with the multi-resistant Camp strain, are given in the Table. Effect of sulphalene in treatment of P. fakiparum ,

Dose, grammes

Days given

Patients studied

Recurrence

Camp strain I.0

1

9 Uganda I strain

1 ’

0

1.0

1

5

3

2-5

1

6

3

Uganda I (py.resist.) strain 1.0

1

11

2*

Another unusual feature was the long time lapse when recrudescence did occur days. The average time between treatment and recrudescence from the normal Uganda strain has been 21 days, and never longer than 28 days for any other antimalarial drug. As support for the association between “pyrimethamine resistance and sulphalene sensitivity”, we report an additional experiment. In the figure the relation between the response to the two drugs is shown in a volunteer in whom the pyrimethamine-resistant strain was losing its resistance. In this substrain there is now a loss of sulphalene sensitivity. This loss of pyrimethamine resistance is relatively characteristic for fleldinduced resistance in strains which are not also chloroquine-resistant (BRAY, 1955). When pyrimethamine resistance is associated with chloroquine resistance, it is usually stable. (33-47)

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Effect of susceptibility to sulphalene by the decline and restoration of pyrimethamine resistance of a P. falciparum strain. # 226 etc. = Chart number of infected volunteer; S = sulphalene; Py = pyrimethamine; Dose in grammes. The parasite studied further.

population which failed to show a change in sulphalene sensitivity was This nonulation came from volunteer indicated by an * (asterisk) in

the Table. The top line shows the time in days. From this original volunteer (l,OOl), 3 sets of subinoculations were made at davs 13, 39. and 59. The drug. resnonse of each aroun of recipients is shown and may be s&m&&d as follows: Group l-2 men were boih cured by 0 * 1 gramme pyrimethamine, indicating a relative loss of pyrimethamine resistance. This explains, we believe, the failure of sulphalene 1 *O gramme shown in the Table * to cure the donor. Group 2-2 patients were inoculated after the donor had demonstrated resistance to 25 mg. pyrimethamine. This strain was still resistant to 1 gramme sulphalene. Group 3-After the donor’s malaria had increased resistance to 100 mg. pyrimethamine, 4 recipients of this parasite population were cured by 1 gramme sulphalene. Discussion The normal Uganda P. falczparum changes its responsiveness to sulphalene after the parasites have been made resistant to pyrimethamine. This is a relatively unusual phenomenon, but it has been reported by GREENBERG (1948) for proguanil-resistant P. gallinaceum. It suggests that the pyrimethamine-chloroquine-resistant strain, Malayan Camp strain, may have developed its sensitivity to sulphalene by the same mechanism. The high degree of sensitivity to sulphalene leads us to conclude that the parasites of this strain are dependent on endogenous synthesis of folic acid, rather than on an exogenous supply. The “normal” Uganda strain does not respond as well as the resistant strain to inhibitors of folic acid synthesis, and at least 50% of the infections persisted in spite of large doses of sulphalene. HAWKING and PERRY (1957) reviewed the evidence for the dependence of P. berghei on dietary PABA. Milk contains approximately 47 mg. of PABA per pint (KON and COWIE, 1961), and milk diets have been used to demonstrate the dependence of parasites on exogenous PABA. These studies will be difficult to interpret. In higher animals, including man, folic acid is taken up by the cell and cannot be synthesized. The membranes must then be permeable to folic acid. In general, organisms which synthesize folic acid do not absorb exogenous folic acid. This block to folic acid absorption usually also limits the uptake of inhibitors of folic acid reductases. Most of the known folic acid synthetase systems are in bacteria, but a few are found in

234 ENHANCED SENSITIVITY TO SULPHALENE IN PYRIMETI-IAhUNE RESISTANT P. fdciparum

animal cells. The best documented example of a protozoan which synthesizes folk acid is in Toxoplasma. We suggest that P. falciparum may also be able to synthesize folk acid. Isolation of folic acid synthetase systems has been difficult and when studies are attempted in isolated systems, they give uncertain results. Because of the difficulty in doing in vivo studies with PABA inhibitors, the presence of such a system in a given organism is usually established by the effects of a sulphone or a sulphonamide on the intact organism. From the evidence, the parasites of P. falciparum should occupy an intermediate position between bacteria and higher animals. This is shown by their ability to change their dependence on folk acid synthesis. TRAGER (1957) has suggested that P. Zophurue can synthesize folic acid, establishing this by directly measuring folic acid accumulation in the parasitized blood. HOTCHKISS and EVANS (1960) were able to circumvent the restraining difficulties of working with the isolated synthetase system by producing several strains of bacteria with different degrees of resistance to several sulphonamides. They thus developed indirect evidence that the synthetase system could exist in several forms. It would appear likely from the analogous bacterial systems that one way to circumvent the action of a reductase inhibitor is by changing cell permeability to the inhibitor. In general, this is related to folk acid uptake since both folk acid inhibitors and reductase inhibitors appear to be handled by the same membrane mechanisms. Futhermore, the folk acid transport across membranes depends on whether or not the cell can make folic acid. If the cell makes folk acid, the membrane is impermeable to folk acid, as well as to some reductase inhibitors. These relationships suggest, but do not prove, that the membrane transport of pyrimethamine is related to pyrimethamine resistance. Even though the data on P. fdcipurum available to us contain no exception to this principle of the association of pyrimethamine resistance with sensitivity to sulphonamides, we must realize that other strains of malaria parasites may operate on a different pattern. 2 sulphadiazine-resistant strains of P. bergk were cross-resistant to proguanil (ROLLO, 1951; THURSTON, 1953) and one was cross-resistant to pyrimethamine (THURSTON 1953). Another strain has been reported with cross resistance to pyrimethamine after sulphadiazine resistance (KRISHNASWAMI, 1954). This raises the possibility that different strains of the same species may vary in their cross resistance pattern. The difference in response pattern to protosil reported by HILL and GOODWIN (1937) and MENK and and MOHR (1939) in P. fuhpurum can be explained either by a possible inherent strain difference or by the fact that one group of patients were semi-immune and the other group were non-immune. At any rate, these two studies were carried out before the folk acid inhibitors were available, and they inject a note of caution in our predictions about the behaviour of all strains of P. falciparum. The practical problem then becomes relatively straightforward. Drugs of both types should be explored for better pharmacological properties. Trimethoprim with a capacity to inhibit pyrimethamine-resistant P. falcaparum, would be preferred over pyrimethamine with its record of rapid induction of resistance (BURGESS and YOUNG, 1959; MARTIN and ARNOLD, 1968a) and a relatively great capacity to bind mammalian reductases (BURCHALL et al., 1965). A large field experience will now be necessary to find the answer to the question whether this associated pyrimethamine resistance and sulphalene sensitivity are necessarily linked, or are only occasionally linked. The exception to this rule will be very important for the future of this approach to chemotherapy.

DANIEL

C. MARTIN

AND

JOHN

D. ARNOLD

235

Summary Experiments were conducted with P. falctparum in volunteers concerning the increased sensitivity of the malaria to a sulphonamide (sulphalene) induced by the parasite’s resistance to pyrimethamine (a dihydrofolate inductase inhibitor). The “normal” P. faZctparum strain was relatively resistant to treatment with sulphalene; only 5 of 11 patients were cured by 2.5 grammes. After resistance to pyrimethamine was induced, the strain was much more sensitive to sulphalene: 9 of 11 patients were cured by 1 gramme. Evidence is presented for the postulate that the sulphonamide sensitivity of a strain depends on whether it does or does not synthesize its own folk acid. It is further postulated that P. falciparum is capable of either using exogenous folk acid or of synthesizing it. The parasites probably do not do both at the same time. REFERENCES BASU, P. C., MONDAL, M. M. & CHAKRABARTI, S. C. (1962). Indian J. Malar., 16, 157. Trans. R. Sot. trap. Med. Hyg., 49, 93. BRAY, R. S. (1955). BURGESS, R. W. & YOUNG M. D. (1959). BUZZ. WZd HZth Org., 20, 37. COGGESHALL, L. T., MAIER, J. & BEST, C. A. (1941). J. Amer. med. Ass., 117, 1,077. COXON, R. V. & HAYES, W. (1945). Trans. R. Sot. trap. Med. Hyg., 39, 195. DE GOWIN, R. L., EPPES, R. B., CARSON, P. E. & POWELL, R. D. (1966). WHO/Mall 66.547. (Cyclostyled document). DIAZ DE LEON, A. (1937). Publ. Hlth. Rep., Wash., 52, 1,460. EARLE, W. C., & PEREZ, M. (1932). J. Lab. clin. Med., 17, 1,124. FINDLAY, G. M., MAEGRAITH, B. G., MARKSON, J. L. & HOLDEN, J. R. (1946). Ann. Trap. Med. Parasit., 40, 358. GREENBERG, J. (1949). J. nat. Malar. Sot., 8, 80. HAWKING, F. & TERRY, W. (1957). Z. Tropenmed. Parasit., 8, 151. HILL, R. A. & GOODWIN, M. H., JR. (1937). S. med. J., 30, 1,170. HOTCHKISS, R. D. & EVANS, A. H. (1960). Fed. Proc., 19, 912. KON, S. K. & COWIE, A. T. (1961). Milk: The Mammary Gland and its Secretion. Vol. Academic Press. II. New York and London: KRISHNASWAMI, A. K., PRAKASH, S., & RAMAKRISHNAN, S. P. (1954). Indian J. Malar., 8, 9. MARTIN, D. C. & ARNOLD, J. D. (1967). Trans. R. Sot. trap. Med. Hyg., 61, 331. (1968a). Ibid., 62, 379. Ibid., 62, 810. -: (1968b). MENK, W. & MOHR, W. (1939). Arch. Schz@-u. Tropenhyg., 43, 117. ROLLO, I. M. (1951). Nature, 168, 332. SAINI, M. (1947). Minerva med., 1, 52. THURSTON, J. P. (1953). Parasitology, 43, 246. TRAGER, W. (1957). Acta tropica, 14, 289.