Malaria parasites, unable to synthesize purine de novo, use host-derived hypoxanthine preferentially as purine source. In a previous study (1990. J. Biol. Chem. 265:6562-6568), we noted that xanthine oxidase rapidly and completely depleted hypoxanthine in human erythrocytes, not by crossing the erythrocyte membrane, but rather by creating a concentration gradient which facilitated hypoxanthine efilux. We therefore investigated the ability of xanthine oxidase to inhibit growth of FCR-3, a chloroquine-resistant strain of Plasmodium falciparum in human erythrocytes in vitro. Parasites were cultured in human group O erythrocytes in medium supplemented, as required, with xanthine oxidase or chloroquine. Parasite viability was assessed by uptake of radiolabeled glycine and adenosine triphosphate-derived purine into protein and nucleic acid, respectively, by nucleic acid accumulation, by L-lactate production, and by microscopic appearance. On average, a 90% inhibition of growth was observed after 72 h of incubation in 20 mU/ ml xanthine oxidase. Inhibition was notably greater than that exerted by i0' M chloroquine (< 10%) over a comparable period. The IC5o for xanthine oxidase was estimated at 0.2 mU/ ml, compared to 1.5 X i0' M for chloroquine. Inhibition was completely reversed by excess hypoxanthine, but was unaffected by oxygen radical scavengers, including superoxide dismutase and catalase. The data confirms that a supply of hostderived hypoxanthine is critical for nucleic acid synthesis in P. falciparum, and that depletion of erythrocyte hypoxanthine pools with xanthine oxidase offers a novel approach to treatment of chloroquine-resistant malaria infection in humans. (J.

Two novel derivatives of clofazimine [3‐(p‐chloroanilino)‐10‐(p‐chlorophenyl)‐2,10‐dihydro‐2‐isopropylimino)phenazine] and the tetramethylpiperidine (TMP)‐substituted phenazines, B4119 [3‐(3‐chloro‐4‐fluoroanilino)‐10‐(3‐chloro‐4‐fluorophenyl)‐2,10‐dihydro‐2(2,2,6,6‐tetramethylpiper‐4‐ylimino)phenazine] and B4158 [3‐(4‐isopropylanilino)‐10‐(4‐isopropylphenyl)‐2,10‐dihydro‐2‐(2,2,6,6‐tetramethylpiper‐4‐ylimino)phenazine] (1–8 μM), were evaluated for activity against chloroquin‐, quinine‐, and sulfadoxine/pyrimethamine‐sensitive and ‐resistant strains of Plasmodium falciparum in vitro, as well as for their effects on polymerisation of haeme to β‐hematin. By using microscopic and flow cytometric methods, it was found that B4119 and B4158, but not clofazimine, inhibited the growth of sensitive, as well as resistant strains of P. falciparum with IC50 values between 0.22 and 0.7 μM, indicating lack of cross‐resistance. Augmentation of anti‐plasmodial activity was observed when B4119 and B4158 were used in combination with chloroquin or mefloquine. Inhibition of the growth of P. falciparum was associated with interference with haeme polymerisation to β‐hematin in vitro, while administration of B4119 to P. berghei‐infected mice was accompanied by a significant reduction in parasitemia and improved therapeutic activity was observed when this agent was combined with chloroquin. The data presented in this study demonstrate that the TMP‐substitution at position 2 on the phenazine nucleus of riminophenazines confers anti‐plasmodial activity on these compounds. These may prove to be useful forerunners in the design of novel anti‐plasmodial pharmacologic agents. Drug Dev. Res. 50:195–202, 2000. © 2000 Wiley‐Liss, Inc.

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Two novel derivatives of clofazimine [3-(p-chloroanilino)-10-(p-chlorophenyl)-2,10-dihydro-2-isopropylimino)phenazine] and the tetramethylpiperidine (TMP)-substituted phenazines, B4119 [3-(3-chloro-4-fluoroanilino)-10-(3-chloro-4-fluorophenyl)-2,10-dihydro-2(2,2,6,6-tetramethylpiper-4ylimino)phenazine] and B4158 [3-(4-isopropylanilino)-10-(4-isopropylphenyl)-2,10-dihydro-2-(2,2,6,6tetramethylpiper-4-ylimino)phenazine] (1-8 µM), were evaluated for activity against chloroquin-, quinine-, and sulfadoxine/pyrimethamine-sensitive and -resistant strains of Plasmodium falciparum in vitro, as well as for their effects on polymerisation of haeme to β-hematin. By using microscopic and flow cytometric methods, it was found that B4119 and B4158, but not clofazimine, inhibited the growth of sensitive, as well as resistant strains of P. falciparum with IC 50 values between 0.22 and 0.7 µM, indicating lack of crossresistance. Augmentation of anti-plasmodial activity was observed when B4119 and B4158 were used in combination with chloroquin or mefloquine. Inhibition of the growth of P. falciparum was associated with interference with haeme polymerisation to β-hematin in vitro, while administration of B4119 to P. bergheiinfected mice was accompanied by a significant reduction in parasitemia and improved therapeutic activity was observed when this agent was combined with chloroquin. The data presented in this study demonstrate that the TMP-substitution at position 2 on the phenazine nucleus of riminophenazines confers antiplasmodial activity on these compounds. These may prove to be useful forerunners in the design of novel anti-plasmodial pharmacologic agents. Drug Dev.