**6. Oxidative chemotherapeutic intervention of Trypanosoma infections**

spermidine, also known as trypanothione (Fairlamb et al., 1985). The oxidised form, trypa‐ nothione disulfide, is generated when trypanothione reduces ROS and its reconversion is catalysed by the trypanothione reductase. Antimonials inhibit this enzyme, leading to an ac‐ cumulation of trypanothione disulfide, which subsequently is not accessible for the reduc‐ tion of ROS (Krauth-Siegel and Comini, 2008). The influence of antimonials on the parasite's redox biology has been verified on cellular level by the fact that trivalent antimonials-resist‐ ant parasites display an increased IC50-value for nitric oxide donors such as NaNO2, SNAP, and DETA NONOate compared to antimonial-sensitive strains (Souza et al., 2010; Holzmül‐ ler et al., 2005; Vanaerschot et al., 2010). Whether nitric oxide resistance is due to elevated

trypanothione levels or due to another antioxidant mode of action is not yet clear.

Amphotericin B (Fig. 2), a polyene macrolide, has been employed in the treatment of *Leish‐ mania* since 1960, but just as a second-line drug. This drug exhibits an excellent antileishma‐ nia activity with more than 90% cure rates. Because the pure compound creates severe side effects and requires long-term treatment and extensive monitoring, liposomal application of amphotericin B is used at the moment which results in cure rates of 3–5 days (up to 100%), is convenient for the patient and is less expensive (Gradoni et al., 2008; Manandhar et al., 2008; Sundar et al., 2002; Thakur et al., 1996). The mode of action can be explained based on its chemical structure, polyene macrolide has been shown to bind to ergosterol, one of the main sterols within *Leishmania* membranes. Interference with this molecule results in an increas‐ ing permeability of the cell membrane which leads to the parasite's death (Balana-Fouce et al., 1998; Amato et al., 2008). Additionally there is some evidence that amphotericin B has an effect on the oxidative response of macrophages (Mukherjee et al., 2010), however further

Miltefosine (hexadecylphosphocholine) is the first and currently the only, orally adminis‐ tered antileishmanial drug (Fig. 2). However, despite cure rates of up to 98% (Roberts, 2006), the drug reveals serious side effects such as vomiting, diarrhea and can cause abnormal physiological development of the foetus. Furthermore, the drug has a relatively long halflife of about 150 hours (Seifert et al., 2007; Maltezou, 2010) which could lead to the develop‐ ment of rapid resistance. Related to its structure, the drug possibly interferes with membranes and membrane-linked enzymes. Currently no verified implications of the drug within the redox biology of the parasite have been found (Rakotomanga et al., 2004; Saint-

**4. Amphotericin B**

230 Drug Discovery

**5. Miltefosine**

Pierre-Chazalet et al., 2009).

experiments are required to verify this effect.

*Trypanosoma* infections, caused by the flagellate protozoan *Trypanosoma* are responsible for a high degree of health problems in endemic countries. They can be divided into two types of pathogens: *Trypanosoma cruzi*, the causative agent of Chagas disease, also known as American trypanosomiasis, since it occurs in Latin America and *Trypanosoma brucei ssp*., the causative agent of sleeping sickness, or human African trypanosomiasis, since it is endemic to sub-Saharan Africa. The current medication is known for its toxici‐ ty, poor activity in immune-suppressed patients and long-term treatment combined with high costs. Moreover, vaccines are not foreseeable in the near future. The *T. cruzi* life cy‐ cle includes three fundamental forms characterized by the relative positions of the flagel‐ lum, kinetoplast and nucleus: Trypomastigotes, epimastigotes and amastigotes, the latter one characterized by their proliferation in any nucleated cell (Prata, 2001). On the one hand Chagas' disease is controlled through elimination of its vectors by using insecti‐ cides and on the other side by chemotherapy. Currently, the drugs used are nifurtimox (4[(5-nitrofurfurylidene)amino]-3-methylthiomorpholine-1,1-dioxide), derived from nitro‐ furan, and benznidazole (*N-*benzyl-2-nitroimidazole-1-acetamide), a nitroimidazole deriv‐ ative. Nifurtimox and benznidazole (Fig. 2) are trypanocidal to all forms of the parasite (Rodriques Coura and de Castro, 2002). However, severe side effects and toxicity have been observed (Kirchhoff, 2000). In addition, there are also reports of mutagenesis result‐ ing in DNA damage (Zahoor et al., 1987). An additional aspect that complicates treat‐ ment is the different susceptibility of different parasite strains to the applied chemotherapeutics (Filardi and Brener, 1987). The mode of action of nifurtimox and benznidazole (Fig. 2) is via the formation of free radicals and/or charged metabolites. The nitro group of both drugs is reduced to an amino group by the catalysis of nitro-re‐ ductases, leading to the formation of various free radical intermediates. Cytochrome P450-related nitro-reductases initiate this process by producing a nitro anion radical (Moreno et al., 1982). Subsequently, the radical reacts with oxygen, which regenerates the drug (Mason and Holtzman, 1975). For example, nifurtimox-derived free radicals may undergo redox cycling with O2, thereby producing H2O2 in a reaction catalysed by the SOD (Temperton et al., 1998). Furthermore, in the presence of Fe3+ the highly reac‐ tive ‧ OH is also being formed according to the Haber-Weiss reaction. These free radicals can subsequently bind to cellular macromolecules such as lipids, proteins and DNA, re‐ sulting in severe damage of parasitic cells (Díaz de Toranzo et al., 1988). In contrast, the trypanocidal effect of benznidazole does not depend on ROS but it is likely that reduced metabolites of benznidazole are covalently binding to cellular macromolecules, thereby revealing their trypanocidal effect (Díaz de Toranzo et al., 1988; Maya et al., 2004). Addi‐ tionally, it has been demonstrated that benznidazole inhibits the *T. cruzi* NADH-fuma‐ rate reductase (Turrens et al., 1996).
