Biochemical Pharmacology, Vol. 36, No. 1, pp. 123-129, 1987. Printed in Great Britain. cm- 295@ 7 s3.00 + 0.00 Pergamon Journals Ltd. zyxwvut ON THE MODE OF ACTION OF PHLORIZIN AS AN ANTIMALARIAL AGENT IN IN VITRO CULTURES OF P~A~~~~~~~ ~A~C~PA~~~ SHIRLEY KUTNER, WILLIAM V. BREUER,* HAGAI GINSBURG and 2;. IOAV CABANTCHIK? Department of Biological Chemistry, Institute of Life Sciences, Hebrew University, JerusaIem 91904, Israel (Received 19 May 1986; accepted 1 August 1986) Abstract-Phlorizin (phloretin-2-j?-glucoside) is a drug which effectively inhibits intraerythrocytic malaria growth in in vitro cultures of Plasmodiumfalciparum IC 5O = 16 -C 7 ,&I). Work with synchron- ously grown cultures indicates that susceptibility to phlorizin is apparent at the trophozoite stage and onward, and that 2-8 hours exposure to the drug causes an irreversible arrest of parasite growth. The drug has also been found to inhibit pores which are induced by the parasite in the host cell membrane (xc% = 17 + 2 @I) and which are apparently essential for intrae~thro~ic growth. The effect on the pores is apparent soon after exposure of the cells to the drug and can be reversed, although extensive washing and incubation in culture conditions are required to achieve it. The results of this study indicate that the putative site of action of phlorizin on the pores is on the cytoplasmic surface of the host cell membrane. The drug which normally cannot permeate uninfected red cells, gains access to the cytoplasm via the pores, appearing in the host cell membrane. Those become eventually the target of phlorizin itself. The proposed mechanism of action of phlorizin on malarial growth invokes blockade of the pores, although additional effects of the drug on intraerythrocytic parasites cannot be ruled out. In the course of intraerythrocytic growth of P. ful- ciparum, the parasite induces marked permeability changes in the host cell membrane fl-51. Those appear as membrane pores through which anions (1,2] and non-electrolytes [3,4] of discrete size can gain either entry into or exit from the red cell cytosol. The pores are detected as early as 6 hr after invasion of red cells, they increase in number with intra- erythrocytic development and are apparently depen- dent on parasite protein synthesis [2]. On the basis of the above-mentioned and other studies, it was suggested that the pores subserve parasite development by providing alternative or supplementary routes of passage of vital materials such as carbohydrates, amino acids and carboxylic acids. So as to test this hypothesis we carried out the present study which is based on the use of phloretin- 2-j3-glucoside (phlorizin), an agent which we have previously shown to effectively suppress malaria growth in in vitro conditions 161. We show here that the phlorizin effect is best exerted at the trophozoite stage and onward and that it is correlated with its inhibitory action on the passage of solutes across the membrane pores induced in the host cell membrane by the intracellular parasites. MATERIALSAND METHODS Chemicals Phlorizin and phloretin were purchased from Sigma, RPMI-1640 from Gibco and [l”C] sorbitol * Present address: Dept of Cell Biology, Hospital for Sick Children Research Institute, Toronto, Ont., Canada MS6 1X8. f To whom correspondence shoufd be addressed. (0.5 Ci/mmol) from the Radiochemical Centre (Amersham, U.K.). [3H]Phlorizin (5 Ci/mmol and 25 Ci/mmol) were obtained as gifts from Prof. G. Semenza (ETH, Zurich) and Dr A Moran (AFRI, Bethesda, MD), respectively. All other chemicals were of best available grade. Cultures The strain of P. faiciparum FCRsTC (a gift from Dr J. B. Jensen) was grown ip 75 cm2 culture flasks (Nunc) using human erythrocytes (O+ or A+, at 2- 2.5% hematocrit) cultured in RPMI-1640 sup- plemented with 25 mM HEPES, 1 mM inosine, 32mM NaHCO? and 10% (v/v) heat-inactivated human plasma (usually AB*). The growth medium was replaced daily and gassed with a mixture of !X.l%Nz, 5%CC&, S%02. Cultures were harvested every 2-4 days when parasitemia (P) reached IO- 20% (P = % infected cells, usually determined microscopically on Giemsa stained thin blood smears). Synchronization of growth was accomplished by two successive treatments of cul- tured cells with 5% mannitol as previously described [7]. Enrichment of infected cells was performed by the sorbitol-Percoll-centrifugation method [2] for isolating rings and by the gelatin-flotation method [8] for isolating trophozoites and schizonts. Effect of inhibitor on parasite development Cells synchronized as shown above were cultured up to the schizont stage, washed aseptically with growth medium, resuspended to 2% hematoc~t and distributed at 150 @ aliquots into wells of a 96 well microtiter plate. Phlorizin was added to 100 ,& final 123