Leischemania
N-Chlorotaurine
- Shows high in vitro activity against promastigotes and amastigotes of Leishmania species
- Causative agents of life-threatening visceral as well as cutaneous and mucocutaneous leishmaniasis. First-line drugs are antimonials, but toxicity and resistance in some endemic areas cause serious problems. In the current study, the antileishmanial activity of the weak oxidant N-chlorotaurine (NCT) was investigated. NCT is a derivative of the amino acid taurine produced by granulocytes and monocytes during oxidative burst, but can also be synthesized chemically and used topically as an antiseptic at a concentration of 1 % (55 mM) in vivo. NCT susceptibility tests were performed in vitro with promastigotes and amastigotes of Leishmania infantum and Leishmania donovani. As NH4Cl is known to increase the activity of NCT by the formation of monochloramine (NH2Cl), co-treatment assays were included in the study. Mean EC50 values after 1 h of treatment were 5.94 mM for L. infantum and 9.8 mM for L. donovani promastigotes. Co-treatment with 5.5 mM NCT plus 19 mM NH4Cl led to complete killing of promastigotes of both strains within 15 min. Amastigotes were inactivated by treatment with 2 mM NCT alone. The results of this study indicate a high potential of NCT against Leishmania species.
Leishmania species are the causative agents of visceral, cutaneous and mucocutaneous leishmaniasis. These vector-borne infections are endemic in more than 80 countries around the world, and according to the World Health Organization they have become the second highest cause of death worldwide after malaria among parasitic diseases, with a 42-fold increase within the last 15 years. Two million new cases – 1.5 million from cutaneous leishmaniosis and 500 000 from the visceral form – occur every year, but as registration of the diseases is compulsory in only 32 countries, a substantial number of cases are never recorded (Singh et al., 2006). Furthermore, co-infection with human immunodeficiency virus presents a serious problem concerning non-responsiveness to therapy and the fact that in AIDS patients almost every strain of Leishmania species can cause any clinical picture mentioned above (Hofman et al., 2000; Mathur et al., 2006).
For therapy, pentavalent antimonials have been successfully administered since 1912, but although these drugs still are the first-line drugs in many countries, they require a prolonged series (up to 30 days) of daily intravenous infusions and they exhibit toxicity (Collin et al., 2004). Moreover, miscellaneous modes of resistance against antimonials are increasing (Ashutosh et al., 2007). A recent study has shown that, in Bihar (India), 60 % of patients currently suffering from leishmaniasis are infected with non-responding Leishmania species (Croft et al., 2006); in 2000, this percentage was around 40 % (Eibl, 2000). Amphotericin B and its liposomal form, as well as miltefosine, are alternative drugs. However, these drugs also have shortcomings including toxicity, cost and possible resistance (Escobar et al., 2002; Sundar & Rai, 2005). The limited therapeutic options, the toxicity of antileishmanial drugs and the increase in resistance thus necessitate a change in the treatment policy.
The aim of the present study was to evaluate the susceptibility of Leishmania infantum and Leishmania donovani promastigotes and amastigotes to N-chlorotaurine (NCT; synthesized chemically as the water-soluble crystalline sodium salt Cl-HN-CH2-CH2-SO3Na, molecular mass 181.57 g; Gottardi & Nagl, 2002). In aqueous solution, NCT can be stored at 2–4 °C for 1 year (Gottardi & Nagl, 2002). As an antiseptic, it is possible that it could be used as a treatment for cutaneous forms of leishmaniasis. NCT is also a natural long-lived oxidant produced by human granulocytes and monocytes from hypochlorite (HOCl) and taurine during oxidative burst. In vivo, NCT is involved in the downregulation of pro-inflammatory cytokines, but its complete function has not yet been entirely elucidated. The ability of human tissues to tolerate synthetically produced NCT is exceptionally high. The human eye, a highly sensitive tissue, has been shown to tolerate 55 mM NCT in a clinical phase II study (Teuchner et al., 2005). Human skin and mucosa also tolerate 55 mM NCT without any noticeable adverse effects (Hofer et al., 2003; Nagl et al., 1998c, 2003). In vitro antimicrobial activity has been reported at micro- to millimolar concentrations against viruses (Nagl et al., 1998a), bacteria (Nagl et al., 2000), fungi (Reeves et al., 2006), helminths (Yazdanbakhsh et al., 1987) and recently against protozoa, i.e. Acanthamoeba species (Fürnkranz et al., 2008) and Trichomonas vaginalis (U. Fürnkranz, M. Nagl, W. Gottardi, M. Duchene, H. Aspöck and J. Walochnik, unpublished results). An enhancing effect of NH4Cl on the efficacy of NCT, explained by the formation of the lipophilic and highly microbicidal monochloramine (NH2Cl) in equilibrium (NCT+NH4Cl-taurine+NH2Cl+H++Cl-), has been outlined previously (Gottardi et al., 2007; Nagl & Gottardi, 1996). Respective tests were included in this study.
In the present study, NCT was shown to be highly effective against Leishmania species. Effective concentrations of NCT were in the millimolar range, which is relatively high compared with other substances used for antileishmanial treatment. However, NCT is an endogenous substance and even the highest concentrations tested in this study are well tolerated by human tissues after topical application (Nagl et al., 1998b, c, 2003; Teuchner et al., 2005). Promastigotes and amastigotes of both strains tested were susceptible to NCT, with L. infantum more susceptible than L. donovani and amastigotes more susceptible than promastigotes. The effectiveness of NCT was higher in PBS than in culture medium, and co-treatment with NH4Cl enhanced the effectiveness of NCT.
Susceptibility of promastigotesThe lowest effective concentration against promastigotes was 2 mM NCT, resulting in growth inhibition in both L. infantum and L. donovani. Mean EC50 values after 1 h of treatment were 5.94±4.07 mM for L. infantum and 9.80±1.30 mM for L. donovani (Table 1T1). Generally, at lower overall concentrations of NCT, L. donovani promastigotes were significantly less susceptible than the promastigotes of L. infantum. Such differences in drug susceptibility between relatively closely related species have been reported previously (Escobar et al., 2002; Yardley et al., 2005) and, in the genus Leishmania, have been explained by variations in sterol and lipid content in the cell membrane (Beach et al., 1979). This may also be the reason for the observed differences in susceptibility to NCT. As NCT possesses a rather non-specific mode of action, which is based on the oxidation of thio, amino and aromatic residues, oxidation of the surface of the parasites can be assumed to depend on the number of residues in the membrane. However, killing of microbes by NCT does not occur by simple oxidation of the surface; it also requires penetration of the oxidant (Gottardi & Nagl, 2005). Treatment with 55 mM NCT led to 100 % lysis of promastigotes of both species investigated within 3 h (data not shown).
Susceptibility of amastigotesAmastigotes were generally more susceptible to treatment than promastigotes. Treatment with 2 mM NCT for 3 h was highly effective, shown by the failure of treated cells to transform into promastigotes, or by reduced transformation in the case of L. donovani (Table 2T2). This phenomenon was also confirmed by a 1 week follow-up, thus also indicating a higher survival rate of amastigotes of L. donovani than those of L. infantum. Untreated controls transformed into promastigotes within 24 h after transfer to culture medium and proliferated within the next 72 h in both species. As THP-1 cells are able to tolerate 2 mM NCT for 24 h, as confirmed previously, we also treated the intracellular amastigotes for 24 h. The 24 h treatment with 2 mM NCT completely inhibited the later transformation into promastigotes, thus indicating complete inactivation of amastigotes in both strains. However, as described above, in culture medium the oxidative capacity of NCT is reduced by over 90 % within 2 h, and thus complete killing of amastigotes cannot be assumed to be solely due to NCT. These late effects might be due to a destructive effect of NCT on factors essential for the parasites, to enhanced killing activity of the host cells or to toxic decay produced by dying or dead amastigotes.
THP-1 monocytes and also macrophages obtained by phorbol myristate acetate stimulation of the monocytes tolerated 2 mM NCT. Although the cells rounded up, they were still viable, as confirmed by trypan blue staining. This result is in agreement with the findings of Park et al. (1995), who showed tolerability to 1.5 mM NCT in the macrophage cell line RAW 264.7. As is generally found for antiseptics, concentrations of NCT tolerated by human cells in vitro, such as human epidermoid carcinoma cells, synoviocytes, and macrophages and neutrophils, are markedly lower (i.e. approximately 0.5–1.5 mM for NCT; Hofer et al., 2003; Kontny et al., 1999; Marcinkiewicz et al., 1995; Nagl et al., 2003; Park et al., 1995) than the concentrations tolerated by the same cell types in the united cell structure in vivo (Nagl et al., 1998c, 2003).
Synergistic effects of NH4ClThe addition of NH4Cl led to enhancement of the efficacy of NCT. The highest level of effectiveness was achieved by co-treatment with 5.5 mM NCT plus 19 mM NH4Cl (molar ratio 1 : 3.4) in PBS, which led to complete killing of promastigotes of both strains within 15 min (Fig. 1F1). These findings are in good accordance with previous studies, where the addition of NH4Cl decreased the killing times of NCT markedly against bacteria and particularly fungi (Gottardi et al., 2007; Nagl & Gottardi, 1996; Nagl et al., 2001). The enhancing effects are explained by the transfer of active chlorine from NCT to NH4Cl, whereby NH2Cl is formed in equilibrium, which penetrates and kills pathogens more rapidly. Enhanced killing effects were higher in PBS than in culture medium. In culture medium, they were only observed for L. infantum, where co-treatment of promastigotes with NCT plus NH4Cl led to a decrease in EC50 and EC90 values after 1 h compared with treatment with NCT alone (Table 1T1). These results are in keeping with the inhibitory effects of high-molecular-mass organic material, such as albumin, on the microbicidal potential of NCT (Nagl & Gottardi, 1996). Some ingredients of the culture medium, mainly thio groups and amino groups of high-molecular-mass proteins, for example from serum contents, partially inactivate NCT (Gottardi et al., 2001). This loss of activity in the medium used in the current study sufficiently explains the absence of significant killing activity of NCT at concentrations of 10 mM or lower. It also explains the lower effects of NH4Cl on the effectiveness of NCT in culture medium compared with PBS. Nevertheless, 100 % inactivation of promastigotes was achieved within 3 h with 55 mM NCT at 25 °C, indicating that the remaining oxidative capacity after 2 h is sufficient to complete the killing of Leishmania species in culture medium.
In conclusion, this study demonstrated the high in vitro activity of NCT against both L. infantum and L. donovani promastigotes and amastigotes, with co-treatment with NH4Cl leading to a further enhancement of effectiveness. NCT is unlikely to provoke resistance, as the mechanism of action is non-specific (Arnitz et al., 2006; Gottardi & Nagl, 2002). Moreover, it is an endogenous amino acid derivative of low molecular mass; thus allergic reactions are unlikely to occur. Further studies to investigate the suitability of NCT for treatment of the cutaneous forms of leishmaniasis would appear to be justified.
