One route for the discovery of new anti-sleeping sickness drugs is the screening of existing drugs for trypanocidal activities [6]. For example, agents that have been developed as potential anti-cancer drugs could also be of use against human African trypanosomiasis as has been shown for the ornithine decarboxylase inhibitor eflornithine [7]. Proteasome inhibitors represent a new class of anti-cancer drugs and have recently been shown to display promising anti-trypanosomal activities [8-12]. In this study, we investigated the effect of the proteasome inhibitor bortezomib (Fig. (Fig.1)1) on bloodstream forms of T. brucei. Bortezomib (Velcade^®) is a novel chemotherapeutic agent for multiple myeloma and mantle cell lymphoma, and was approved by the FDA for treatment of these malignancies in 2003 and 2006, respectively [13].

The trypanocidal activity of bortezomib was determined with T. brucei bloodstream forms 427-221a while the general cytotoxicity of the drug was evaluated with human myeloid leukaemia HL-60 cells. The tests were analysed by the Alamar Blue assay as described previously [14]. In brief, cells were seeded in 24-well plates in a final volume of 1 ml of appropriate culture medium (trypanosomes: Baltz medium plus 16.7% heat-inactivated foetal bovine serum [15]; HL-60 cells: RPMI 1640 plus 16.7% heat-inactivated foetal bovine serum) containing various concentration of bortezomib and 1% DMSO. Wells containing medium and 1% DMSO served as controls. The initial densities were 10⁴/ml for trypanosomes and 10⁵/ml for HL-60 cells. After 24 h incubation, 100 μl Alamar Blue (11.11 mg resazurin sodium salt in 100 ml PBS) was added and the cells were incubated for a further 48 h so that the total incubation time was 72 h. Then, the plates were read on a microplate reader using a test wavelength of 570 nm and a reference wavelength of 630 nm. The 50% growth inhibition (GI₅₀) values, i.e. the concentration of the drug necessary to reduce the growth rate of trypanosomes and HL-60 cells by 50% to that of controls, was determined by linear interpolation according to the method described in [16]. The minimum inhibitory concentration (MIC) values, i.e. the concentration of the drug at which all trypanosomes and HL-60 cells were killed, was determined microscopically.

Bortezomib showed a dose-dependent effect on the growth of T. brucei bloodstream forms with a GI₅₀ value of 3.3 nM and a MIC value of 10 nM (Fig. (Fig.2).2). For comparison, the trypanocidal activity of the commercial anti-sleeping sickness drug melarsoprol was determined under the same experimental conditions. Melarsoprol exhibited a GI₅₀ value of 3.8 nM and a MIC value of 100 nM (Fig. (Fig.2).2). Thus, the trypanocidal activity of bortezomib was similar to melarsoprol with respect to the GI₅₀ value and was 10 times greater with respect to the MIC value. As can be expected from a drug to treat hematologic malignancies, bortezomib exhibited also cytotoxic activity against HL-60 cells with a GI₅₀ value of 26.7 nM and a MIC value of 100 nM (Fig. (Fig.2).2). The GI₅₀ value of bortezomib for HL-60 cells is within the range of the average GI₅₀ value of 7 nM for the drug across 60 tumour cell lines (including HL-60 cells) [17]. Melarsoprol, on the other hand, was much less toxic to HL-60 cells, displaying a GI₅₀ value of 3260 nM and a MIC value of 10,000 nM (Fig. (Fig.2).2). As a result, the GI₅₀ and MIC ratios of cytotoxic/trypanocidal activities (selectivity indices) for bortezomib were less favourable (GI₅₀ ratio = 8; MIC ratio = 10) than that of the anti-sleeping sickness drug melarsoprol (GI₅₀ ratio = 860; MIC ratio = 100).

To check whether the trypanocidal activity of bortezomib is due to inhibition of the proteasome, the activity of this multi-enzyme complex in trypanosomes exposed to the drug was determined. In this experiment, 10⁷ trypanosomes/ml were incubated with or without 100 μM bortezomib for 2 h. After washing three times with PBS/1% glucose, the trypanosomes were lysed in 10 mM Tris, 2 mM ATP, 0.1 mM EDTA, pH 7, 1 mM DTT, 0.2% NP-40 and centrifuged at 16000 × g. The chymotrypsin-like activity and the trypsin-like activity of the clarified supernatant were assayed in 50 mM HEPES, pH 7.5 with 10 μM Suc-LLVY-AMC and 10 μM Z-GGR-AMC, respectively. Surprisingly, bortezomib inhibited the chymotrypsin-like activity and the trypsin-like activity of the proteasome in trypanosomes only by 30% and 10%, respectively. Under the same experimental conditions, the drug inhibited the proteasomal chymotrypsin-like and trypsin-like activity in HL-60 cells by 100% and 90%, respectively. These findings indicate that the trypanocidal action of bortezomib is most likely not the result from inactivation of the proteasome.

For treatment of multiple myeloma, the recommended dose and treatment schedule of bortezomib is 1.3 mg/m² administered as a 3 to 5 second bolus intravenous injection on days 1, 4, 8 and 11 of a three week cycle, for up to 8 cycles [18]. Pharmacokinetic/pharmacodynamic studies showed that on day 11 the mean plasma concentration of bortezomib falls from 422 nM to 10 nM within 16 h of administration of the drug [19]. As 10 nM is the MIC value of bortezomib, we wanted to investigate whether exposure of trypanosomes to 10 nM of the drug for 16 h is long enough to kill the parasites. To this end, 5 × 10⁵ trypanosomes/ml were treated with or without 10 nM bortezomib for 16 h, then diluted to 10⁴ cells/ml in fresh medium for further incubation and counted every 24 h using a Neubauer haemocytometer. For the first 48 h of re-incubation, the densities of cultures containing trypanosomes that had been exposed to bortezomib decreased continuously (Fig. (Fig.3).3). Thereafter, the numbers of trypanosomes started to increase but did not reach those of control cultures (Fig. (Fig.3).3). This result shows that treatment of trypanosomes with 10 nM bortezomib for 16 h leads in the killing of 90% of the parasites.