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In recent years there has been a major resurgence of African sleeping sickness, with an estimated 300,000 people affected (21). In humans, the disease is caused by infection with the tsetse fly-transmitted protozoan parasites Trypanosoma brucei gambiense (western and central Africa) and Trypanosoma brucei rhodesiense (eastern and southern Africa). Untreated, sleeping sickness is fatal. Current chemotherapeutic regimes are unsatisfactory (7, 15); they require hospitalization and are expensive, can fail to eradicate parasitemia, and often produce toxic side effects. For example, melarsoprol is the only effective drug for the advanced stage of sleeping sickness, which occurs once parasites have invaded the central nervous system. However, melarsoprol treatment can cause arsenic encepalopathy and results in 5 to 10% patient mortality. Consequently, the development of new trypanocidal drugs is a major priority of the World Health Organization (WHO).

In the course of work aimed at expressing modified forms of the viral M2 protein in trypanosomes, we noticed that the anti-influenza virus agent rimantadine (-methyl-1-adamantane methylamine hydrochloride) was highly effective at killing bloodstream forms of T. brucei in vitro. Overnight incubation in medium containing more than 5 µg ml¹ resulted in the death of all cells in the culture. Rimantidine is an amide derivative of amantadine, and both drugs are licensed for the treatment and prophylaxis of influenza A (5). To establish the extent of this activity, bloodstream form T. brucei (strain 427) was cultured in the presence of rimantadine or amantadine, and the concentrations of the drugs which inhibited growth by 50% (IC₅₀) and 90% (IC₉₀) were determined. Both compounds were found to have trypanocidal activity, with rimantadine being more effective (Table 1). In these experiments, the cells were incubated for 3 days at 37°C in 4-ml volumes of modified Iscove's medium (pH 7.4) (10) in 25-cm³ flasks. The density of untreated cultures increased from 1 × 10⁵ cells ml¹ to 4 × 10⁶ cells ml¹ under these conditions. We next investigated the effect of pH on rimantadine activity and found that above neutral it had increased toxicity (Fig. 1A and Table 1). For example, 2 µg of rimantadine ml¹ inhibited cell growth by 70% at pH 7.4 (the normal pH of blood), whereas at pH 7.0, T. brucei grew at a similar rate with or without the drug (Fig. 1A). At higher drug concentrations (20 µg ml¹) rimantadine was toxic at all pH levels tested (Fig. 1A). Amantadine was also found to exhibit activity, but only at concentrations of the drug that are not physiologically attainable. As with rimantadine, the trypanocidal effect was pH dependent and was enhanced in an alkaline environment (Table 1).

View larger version (13K): [in this window] [in a new window]   FIG. 1.   Susceptibility of bloodstream form T. brucei (A), T. cruzi epimastigotes (B), and L. major promastigotes (C) to rimantadine at different pHs. Parasites were cultured as described in the text in the presence or absence of 2 µg of rimantadine ml1 (A) or 20 µg of rimantadine ml1 (B, C). In the case of T. brucei, treatment with 20 µg of rimantadine ml1 resulted in total cell death at each pH tested. Values are expressed as a percentage of growth obtained at optimal pH in the absence of drug.

We then determined whether rimantadine was active against the trypanosmatid parasites Trypanosoma cruzi (which causes Chagas' disease, or American trypanosomiasis) and Leishmania major (a causative agent of cutaneous leishmaniasis). The T. cruzi and L. major growth inhibition experiments were carried out in Nunclon 24 well plates in 2 ml of growth medium. In the case of T. cruzi (CL Brener clone) (3), the initial inoculum was 2 × 10⁵ epimastigotes ml¹. After 5 days of incubation at 28°C in RPMI medium (13) in the absence of drug, the cell density increased to 2 × 10⁷ ml¹. With L. major (strain 5ASKH), 2 × 10⁵ promastigotes ml¹ were incubated in minimal essential medium (6) at 24°C. Under these conditions, the growth rate was similar to that of T. cruzi. Both parasites were found to be susceptible to rimantadine, although to a lesser extent than T. brucei (Fig. 1). At pH 7.8, the IC₅₀s for T. cruzi epimastigotes and L. major promastigotes were 6.0 and 10.5 µg ml¹ respectively. The response of T. cruzi cells to rimantadine treatment was characterized by swelling and loss of typical epimastigote morphology, including the flagellum. T. cruzi and L. major also exhibited pH-dependent sensitivity to rimantadine (Fig. 1B and C). In an alkaline environment they were more susceptible, whereas at an acidic pH they were largely refractory. Consistent with this, rimantadine treatment (20 µg ml¹) of an L. major-infected human macrophage cell line (THP-1) had no effect on parasitemia (data not shown). In mammalian cells the intracellular amastigote form of the parasite resides in the acidic phagolysosme compartment. We also examined the effect of amantadine on T. cruzi. Under normal culture conditions (pH 7.4), there was no significant growth inhibitory effect at concentrations below 20 µg ml¹. However, greater toxicity was observed at higher pH levels, and at pH 8.4 the IC₅₀ was 12 µg ml¹ compared with 1.8 µg ml¹ for rimantadine under the same conditions.

In influenza A virus-infected cells the target of both rimantadine and amantadine is the viral ion channel protein M2 (8, 16). In its tetrameric form, M2 functions by translocating protons into the virus interior (4, 17, 18), an acidification process which facilitates virus uncoating (19). The drugs bind to an amino acid(s) within the NH₂-terminal portion of the M2 transmembrane region, leading to blockage of the proton channel, probably as a result of conformational changes (17). In trypanosomatids, rimantadine toxicity is associated with a reduced ability to tolerate an alkaline environment (Fig. 1). Therefore, one possibility, by analogy with the situation in influenza virus-infected cells, is that rimantadine blocks a transmembrane proton pump which acts to maintain intracellular pH. Proton-translocating, membrane-localized ATPases (H⁺-ATPases) have been identified as the primary mechanism for the maintenance of intracellular pH homeostasis in trypanosomatids (22). The use of genetic (11) and biochemical (2) approaches should make it possible to test if these proton pumps are the target for rimantadine and to investigate the precise mechanism of action.

Rimantadine has many desirable properties as a chemotherapeutic agent. It is inexpensive, can be taken orally, produces fewer side affects than amantadine (9), and readily crosses the blood brain barrier. In addition, the pharmacokinetics have been extensively investigated (20); it is well absorbed from the gastrointestinal tract and in humans has a plasma half-life of 24 to 36 h (1). Serum levels above 1 µg ml¹ have been reported (14). The results presented here suggest that rimantadine may have potential as a drug against African sleeping sickness, particularly if it can be administered in combination with agents which elevate blood pH. Furthermore, the considerable difference between the susceptibilities of T. brucei to amantadine and rimantadine (Table 1) suggests that the trypanocidal effects of other aminoadamantane derivatives (12) warrant investigation.