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Mechanisms of Action: Physiological Effects

The Anti-Influenza Virus Drug Rimantadine Has Trypanocidal Activity

John M. Kelly, Michael A. Miles, Anita C. Skinner
John M. Kelly
Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1E 7HT, and
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Michael A. Miles
Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1E 7HT, and
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Anita C. Skinner
Division of Virology, National Institute for Medical Research, The Ridgeway, London NW7 1AA, United Kingdom
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DOI: 10.1128/AAC.43.4.985
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ABSTRACT

We report here that bloodstream forms of the African trypanosome,Trypanosoma brucei, are sensitive to the anti-influenza virus drug rimantadine (50% inhibitory concentration of 1.26 μg ml−1 at pH 7.4). The activity is pH dependent and is consistent with a mechanism involving inhibition of the ability to regulate internal pH. Rimantadine is also toxic to the trypanosomatid parasites Trypanosoma cruzi and Leishmania major.

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 parasitesTrypanosoma 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 (α-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 ml−1 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 (Table1). 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 × 105cells ml−1 to 4 × 106 cells ml−1 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 Table1). For example, 2 μg of rimantadine ml−1 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 ml−1) 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).

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Table 1.

Susceptibilities of the cultured bloodstream form ofT. brucei to rimantadine and amantadine

Fig. 1.
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Fig. 1.

Susceptibility of bloodstream form T. brucei (A), T. cruzi epimastigotes (B), andL. 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 ml−1 (A) or 20 μg of rimantadine ml−1 (B, C). In the case of T. brucei, treatment with 20 μg of rimantadine ml−1resulted 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) andLeishmania 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 ml−1. After 5 days of incubation at 28°C in RPMI medium (13) in the absence of drug, the cell density increased to 2 × 107ml−1. With L. major (strain 5ASKH), 2 × 105 promastigotes ml−1 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. cruziepimastigotes and L. major promastigotes were 6.0 and 10.5 μg ml−1 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 ml−1) 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 ml−1. However, greater toxicity was observed at higher pH levels, and at pH 8.4 the IC50 was 12 μg ml−1 compared with 1.8 μg ml−1 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 ml−1have 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.

ACKNOWLEDGMENTS

This work was funded by the Leverhulme Trust.

FOOTNOTES

    • Received 24 November 1998.
    • Accepted 27 January 1999.
  • Copyright © 1999 American Society for Microbiology

REFERENCES

  1. 1.↵
    1. Anderson E. L.,
    2. Van Voris L. P.,
    3. Bartram J.,
    4. Hoffman H. E.,
    5. Belshe R. B.
    Pharmacokinetics of a single dose of rimantadine in young adults and children. Antimicrob. Agents Chemother. 31 1987 1140 1142
    OpenUrlAbstract/FREE Full Text
  2. 2.↵
    1. Benchimol F.,
    2. De Souza W.,
    3. Vanderheyden N.,
    4. Zhong L.,
    5. Lu H.-G.,
    6. Moreno S. N. J.,
    7. Docampo R.
    Functional expression of a vacuolar-type H+-ATPase in the plasma membrane and intracellular vacuoles of Trypanosoma cruzi. Biochem. J. 332 1998 695 702
    OpenUrlAbstract/FREE Full Text
  3. 3.↵
    1. Cano M. I.,
    2. Gruber A.,
    3. Vasquez M.,
    4. Cortes A.,
    5. Levin M. J.,
    6. Gonzalez A.,
    7. Degrave W.,
    8. Rondinelli E.,
    9. Zingales B.,
    10. Ramirez J. L.,
    11. Alonso C.,
    12. Requena J. M.,
    13. Franco da Silveira J.
    Molecular karyotype of clone CL Brener chosen for the Trypanosoma cruzi genome project. Mol. Biochem. Parasitol. 71 1995 273 278
    OpenUrlCrossRefPubMedWeb of Science
  4. 4.↵
    1. Chizhmakov I. V.,
    2. Geraghty F. M.,
    3. Ogden D. C.,
    4. Hayhurst A.,
    5. Antoniou M.,
    6. Hay A. J.
    Selective proton permeability and pH regulation of the influenza virus M2 channel expressed in mouse erythroleukaemia cells. J. Physiol. 494 1996 329 336
    OpenUrlCrossRefPubMedWeb of Science
  5. 5.↵
    1. Ellsworth A. J.,
    2. Witt D. M.,
    3. Dugdale D. C.,
    4. Oliver L. M.
    Mosby’s medical drug reference. 1998 Mosby-Year Book Inc. St. Louis, Mo
  6. 6.↵
    1. Evans D. A.
    In vitro cultivation and biological cloning of Leishmania Protocols in molecular parasitology. Hyde J. E. 1993 29 41 Humana Press Inc. Totowa, N.J
  7. 7.↵
    1. Gompel A. V.,
    2. Vervoort T.
    Chemotherapy for leishmaniasis and trypanosomiasis. Curr. Opin. Infect. Dis. 10 1997 469 474
    OpenUrlCrossRef
  8. 8.↵
    1. Hay A. J.
    The action of adamantanamines against influenza A viruses: inhibition of the M2 ion channel protein. Semin. Virol. 3 1992 21 30
    OpenUrl
  9. 9.↵
    1. Hayden F. G.,
    2. Gwaltney J. M.,
    3. Van de Castle R. L.,
    4. Adams K. F.,
    5. Giordani B.
    Comparative toxicity of amantadine hydrochloride and rimantadine hydrochloride in healthy adults. Antimicrob. Agents Chemother. 19 1981 226 233
    OpenUrlAbstract/FREE Full Text
  10. 10.↵
    1. Hirumi H.,
    2. Hirumi K.
    Continuous culture of Trypanosoma brucei blood stream forms in a medium containing a low concentration of serum protein without feeder cell layers. J. Parasitol. 75 1989 985 989
    OpenUrlCrossRefPubMedWeb of Science
  11. 11.↵
    1. Kelly J. M.,
    2. Taylor M. C.,
    3. Rudenko G.,
    4. Blundell P. A.
    Transfection of the African and American trypanosomes Electroporation protocols for microorganisms. Nickoloff J. A. 1995 349 359 Humana Press Inc. Totowa, N.J
  12. 12.↵
    1. Kolocouris N.,
    2. Kolocouris A.,
    3. Foscolos G. B.,
    4. Fytas G.,
    5. Neyts J.,
    6. Padalko E.,
    7. Balzarini J.,
    8. Snoeck R.,
    9. Andrie G.,
    10. De Clercq E.
    Synthesis and antiviral activity of some new aminoadamantane derivatives. J. Med. Chem. 39 1986 3307 3318
    OpenUrlCrossRef
  13. 13.↵
    1. Miles M. A.
    Culturing and biological cloning of Trypanosoma cruzi Protocols in molecular parasitology. Hyde J. E. 1993 15 28 Humana Press Inc. Totowa, N.J
  14. 14.↵
    1. Patriarca P. A.,
    2. Kater N. A.,
    3. Kendal A. P.,
    4. Bregman D. J.,
    5. Smith J. D.,
    6. Sikes R. K.
    Safety of prolonged administration of rimantadine hydrochloride in the prophylaxis of influenza A virus infections in nursing homes. Antimicrob. Agents Chemother. 26 1984 101 103
    OpenUrlAbstract/FREE Full Text
  15. 15.↵
    1. Pepin J.,
    2. Milford F.
    The treatment of human African trypanosomiasis. Adv. Parasitol. 33 1994 1 47
    OpenUrlCrossRefPubMedWeb of Science
  16. 16.↵
    1. Pinto L. H.,
    2. Holsinger L. J.,
    3. Lamb R. A.
    Influenza virus M2 protein has ion channel activity. Cell 69 1992 517 528
    OpenUrlCrossRefPubMedWeb of Science
  17. 17.↵
    1. Pinto L. H.,
    2. Lamb R. A.
    Understanding the mechanism of action of the anti-influenza virus drug amantadine. Trends Microbiol. 3 1995 271
    OpenUrlCrossRefPubMed
  18. 18.↵
    1. Sugrue R. J.,
    2. Hay A. J.
    Structural characteristics of the M2 protein of influenza A viruses: evidence that it forms a tetrameric channel. Virology 180 1991 617 624
    OpenUrlCrossRefPubMedWeb of Science
  19. 19.↵
    1. Wharton S. A.,
    2. Belshe R. B.,
    3. Skehel J. J.,
    4. Hay A. J.
    The virion M2 protein in influenza virus uncoating: specific reduction in the rate of membrane fusion between virus and liposomes by amantadine. J. Gen. Virol. 75 1994 945 948
    OpenUrlCrossRefPubMedWeb of Science
  20. 20.↵
    1. Wills R. J.,
    2. Farolino D. A.,
    3. Choma N.,
    4. Keigher N.
    Rimantadine pharmacokinetics after single and multiple doses. Antimicrob. Agents Chemother. 31 1987 826 828
    OpenUrlAbstract/FREE Full Text
  21. 21.↵
    World Health Organization Control of tropical diseases: sleeping sickness. 1994 WHO Geneva, Switzerland
  22. 22.↵
    1. Zilberstein D.,
    2. Shapira M.
    The role of pH and temperature in the development of Leishmania parasites. Annu. Rev. Microbiol. 48 1994 449 470
    OpenUrlCrossRefPubMedWeb of Science
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The Anti-Influenza Virus Drug Rimantadine Has Trypanocidal Activity
John M. Kelly, Michael A. Miles, Anita C. Skinner
Antimicrobial Agents and Chemotherapy Apr 1999, 43 (4) 985-987; DOI: 10.1128/AAC.43.4.985

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The Anti-Influenza Virus Drug Rimantadine Has Trypanocidal Activity
John M. Kelly, Michael A. Miles, Anita C. Skinner
Antimicrobial Agents and Chemotherapy Apr 1999, 43 (4) 985-987; DOI: 10.1128/AAC.43.4.985
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KEYWORDS

antiviral agents
Rimantadine
Trypanocidal Agents
Trypanosoma brucei brucei

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