Reviewer 2: +(N+E)

1. The multicenter of N+E combination therapy for second-stage sleeping sickness presented on behalf of G. Priotto and the NECT Study Team offers a better option than Eflornithine alone. In the comparative trial between the two arms, Eflornithine is given at 400mg/kg/day QIDX 14 days and the N+E combination of Nifurtimox at 15 mg/kg/day for 10 days and Eflornithine at 400mg/kg/day BID X 7 days. Thus the reduced dose of Eflornithine (reduced by half per day and for half the duration)can explain that while primary efficacy in one-sided non-inferiority test already show difference in cure rate in favor of N+E and the secondary efficacy indicators are also in favor of N+E, the major adverse events particularly fever, infection and neutropenia aside from selected clinical events (hypertension,diarrhea) are much lower with N+E. The presence of more events with N+E has to be explained.

Additional Related Materials reviewed:

2. Nifurtimox plus Eflornithine for Late-Stage Sleeping Sickness in Uganda: A Case Series (F Checchi et al, Plo Negl Trop Dis 1(2):e64. doi:10.1371/journal.pntd.0000064) A prospective case series of 31 late-stage gambiense sleeping sickness (Human African , HAT) reports efficacy and safety outcomes in patients treated with a combination of nifurtimox and eflornithine in Yumbe,northwest Ugandain 2000-2003, following on a previously reported terminated trial in nearby Omugo, where 17 patients received the combination under the same conditions. Eligible sequential late-stage patients received 400mg/kg/day eflornithine for 7 days plus 15mg/kg/day nifurtimox (20 mg/kg for children <15 years old) for 10 days. Efficacy (primary outcome)was monitored for 24 months post-discharge. Efficacy ranged from 90.3% to 100%; 5patients had major adverse events (neutropenia common, 9/31 patients)

The Conclusions/Significance reads: Combined with the previous group of 17 trial patients, this case series yields a group of 48 patients treated with N+E among whom no deaths judged to be treatment- or HAT-related, no treatment terminations and no relapses have been noted,a very favourable outcome in the context oflate-stage disease. N+E could be the most promising combination regimen available for sleeping sickness, and deserves further evaluation.

3. Nifurtimox-Eflornithine Combination Therapy for Second-Stage Trypanosoma brucei gambiense Sleeping Sickness: A Randomized Clinical Trial in Congo (G Priotto et al, Clinical Infectious Diseases 2007;45:1435-42)

Efficacy and safety compared N+E combination (Nifurtimox 15mg/kg/day given orally every 8 hours for 10 days plus Eflornithine 400mg/kg intravenously every 12 hours for 7 days) with Eflornithine alone given 400 mg/kg per day intravenously every 6 hrs for 14 days, for treatment of second-stage disease in a randomized , open-label, active-control Phase III clinical trial. Patients were observed for 18 months. The study's outcomes were cure and adverse event attributable to treatment. A total of 103 patients were enrolled.Cure rates were similar for both groups (E 94.1% vs N+E 96.2%). Severe reactions were less with the N+E group, 9.6% versus E 25.5%.There were no deaths in the N+E group versus 1 in E. The conclusion was that the N+E combination was promising and if corroborated by ongoing findings from other study sites, it will be a major advance over current therapies.

4. Three drug combinations for late-stage trypanosoma brucei gambiense Sleeping Sickness: A Randomized Clinical Trial in Uganda. G Priotto et al 2006, PloS Clin Trials 1(8): e39. doi:10.1371/journal.pctr.0010039

This was a randomized open-label, active control, parallel clinical trialcompring three arms. Three drug combinations were randomily assigned to patients: -nifurtimox or M+N, melarsoprol- eflornithine or M+E, and nifurtimox-eflornithine or N+E. Dosages were uniform: Intravenous melarsoprol 1.8 mg/kg/day, daily for 10 days; IV eflornithine 400 mg/kg/day, every 6 hrs for 7 days; oral nifurtimox 15mg/kg /day(adults) or 20 mg/kg/day (children <15 years) given every 8 hrs for 10 days. Patients were followed up for 24 months.

Outcomes were cure rates and adverse events attributable to treatment. Result: Randomization performed in 54 patients before enrollment was suspended due to unacceptable toxicity in one of the three arms. Cure rates obtained with ITT analysis were M+N 44.4%, M+E 78.9%, and N+E 94.1%. Significantly higher with N+E (p=0.003) and M+E (p=0.045). Adverse events were less frequent and less severe with N+E, resulting in fewer treatment interruptions and no fatalities. N+E is a promising combination but no conclusion can be drawn from this interrupted study. (See larger current studies above)

Title, Author, Journal Objectives of Design Participants Interventions Outcome Results Conclusi,on/ (year, volpp) the study parameters/En Recommendations dpoints Nifurtimox – Eflornithine To evaluate the Randomized, Inclusion criteria: Eflornithine alone Cure rates and 103 patients with second N+E appears to be a Combination Therapy for efficacy of the open-label, confirmed 2nd stage versus adverse events stage disease were enrolled. promising first line Second Stage T. b. nifurtimox + active-control, T.b.gambiense eflornithine + attributable to Cure rates for eflornithine: therapy for second gambiense Sleeping eflornithine phase III infection nifurtimox. treatment 94.1%, for E+N 96.2%. stage sleeping Sickness: A Randomized combination clinical trial Exclusion criteria: Patients were sickness. Clinical Trial in Congo therapy comparing 2 -Age<15 years old observed for 18 Severe reactions: (Priotto et. al.) arms - months. Eflornithine: 25.5% Clinical Infectious -history of stage 2 E+N: 9.6% Diseases, December 2007, HAT treated during Vol. 45, pp. 1435-1442 the preceding 36 There was one death in the months E arm and no death in the -severe E+N. comorbidities -hemoglobin < 5g/dL -inability to complete 18 months of follow up for other reasons Three Drug Combinations To compare the Randomized, Stage 2 patients 3 drug Cure rates and 54 patients were N+E appears to be a for Late Stage efficacy and open-label, diagnosed in combinations: adverse events randomized. Enrollment was promising first line Trypanosoma brucei safety of 3 drug active control, Northern Uganda Melarsoprol + attributable to suspended because of therapy that may gambiense Sleeping combinations for parallel Inclusion criteria: nifurtimox treatment unacceptable toxicity in one improve treatment Sickness: A Randomized the treatment of clinical trial confirmed 2nd stage (M+N), of the three arms. Cure rates sleeping sickness. Clinical Trial in Uganda late stage human comparing 3 T.b.gambiense melarsoprol + obtained with ITT analysis However, larger (Priotto et. al.) African arms infection eflornithine were M+N 44.4%, M+E studies need to be PLOS Clinical Trials, trypanosomiasis Exclusion criteria: (M+E), 78.9%, N+E 94.1%, and done to evaluate the December 2006, pp. 1-8 cause by -BW<10kg nifurtimox + were significantly higher drug combination. Trypanosoma -pregnancy eflornithine with N+E (p=0.003) and brucei -history of stage 2 (N+E). M+E (p=0.045) than with gambiense HAT treated during Melarsoprol and M+N. the preceding 24 eflornithine were months given IV while Adverse events were less -unlikelihood of nifurtimox was frequent and severe with completing the 2 given orally. N+E, resulting in fewer year follow up Dosages were treatment interruptions and uniform. Patients no fatalities. 4 died who were followed up were taking M+N and one for 24 months. who was taking M+E. Nifurtimox plus To report the Case series Late stage patients Patients received Cure rates and Efficacy ranged from 90.3% Combined with the Eflornithine for Late efficacy and with a confirmed 400 mg/kg/day efficacy were to 100.0% according to previous group of 17 Stage Sleeping Sickness in safety outcomes diagnosis of T.b. eflornithine for 7 monitored for analysis approach. All 31 trial patients, this Uganda from a gambiense infection days plus 15 24 months post patients were discharged case series yielded 48 Title, Author, Journal Objectives of Design Participants Interventions Outcome Results Conclusi,on/ (year, volpp) the study parameters/En Recommendations dpoints Checchi et. al. prospective case Inclusion criteria: mg/kg/day discharge; alive, but 2 died post patients treated with PLOS Neglected Tropical series of 31 late -non pregnant nifurtimox for 10 clinical and discharge of non-HAT and N+E, among whom a Diseases, 2007, Vol 1, stage T.b. -BW>10kg days. Patients laboratory non-treatment causes, one very favorable Issue 2, pp. 1-6 gambiense -late stage T.b. were monitored adverse events was lost to follow-up. outcome after sleeping gambiense HAT for 24 months were monitored treatment was found. sickness treated -no history of HAT post-discharge. during Five patients had major N+E could be the with a treatment in the treatment. adverse events during most promising combination of prior 24 months treatment and neutropenia treatment regimen for eflornithine and -follow up can be was common. sleeping sickness and nifurtimox in insured deserves further Uganda evaluation. The blood – brain barrier To explore the Experimental Murine model of Eflornithine and Effect of drug Eflornithine crosses the Eflornithine crossed significantly limits pharmacokinetic with animal sleeping sickness Eflornithine + on barrier blood-CNS interface by the healthy blood- eflornithine entry into T. characteristics of model other drug integrity diffusionand eflornithine CNS interfaces brucei brucei infected eflornithine combinations to entry into the CNS can be poorly, but this could mouse brain transport across investigate Effect of enhanced with . This be improved by co- (Sanderson et. al.) the healthy transporters parasite on explains the observed administering Journal of blood-CNS barrier synergy of eflornithine and suramin, but not Neurochemistry, 2008, interfaces both integrity/permea suramin combinations in nifrutimox, Vol. 107, pp. 1136 – 1146 alone and with blility CNS efficacy models and is penatmidine or other anti- first to demonstrate that melarsoprol. trymanosomal Effect of drugs combination therapy can drugs on transporters prove efficacious due to enhanced delivery of the To investigate drug to the CNS. the potential removal of The parasites reach the CNS eflornithine by early in the course of the BBB efflux infection, irreversible blood- transporter brain and blood-CSF barrier breakdown is unnecessary To explore for parasites to reach the eflornithine drug CNS. Parasites that cross the delivery and BBB in vivo remain viable, blood-CNS and widespread BBB barrier integrity dysfunction occurs during at set time points the terminal stage of the in mice affected disease. with T.b. brucei and to correlate the parasite existence within the CNS Melarsoprol free drug Editorial Title, Author, Journal Objectives of Design Participants Interventions Outcome Results Conclusi,on/ (year, volpp) the study parameters/En Recommendations dpoints combinations for Second commentary Stage Gambian Sickness: The Way to Go (Chappuis, Francois) Clinical Infectious Diseases, December 2007, Vol. 45, pp. 1435-1442 Innate lack of To Two different All trypanosome stocks DFMO is not an susceptibility of characterized assays were were susceptible to appropriate Ugandan Trypanosoma Trypanosoma used to suramin and nifurtimox. alternative or brucei rhodesiense to brucei determine the Differences in the backup drug for DL-alpha- rhodesiense drug susceptibility to treatment of difluoromethylornithine isolates from susceptibility of melarsoprol were Rhodesian sleeping (DFMO) South East the field observed in the sickness in (Iten, M. et. al.) Uganda for isolates: the [3H]hypoxanthine Uganda. Trop Med Parasitol, susceptibility [3H]hypoxanthi incorporation assay, but 1995, Vol. 46, No. 3, to the drugs ne incorporation could not be confirmed in pp.190-194 suramin, assay (24 hours) the long term viability ... nifurtimox, and the long assay. All T. b. melarsoprol term viability rhodesiense stocks were and DL-alpha- assay (10 days) found in vitro to have difluoromethyl innate tolerance to DFMO, under conditions (DFMO) where T. b. gambiense stocks from West Africa were susceptible to the drug. Ugandan T. b. rhodesiense stocks did respond to 25-100 micrograms/ml after 10 days of drug exposure, but the DFMO level reached in during treatment is only 16.3 +/- 7.8 micrograms/ml High-dose nifurtimox To determine Open Trial Thirty patients Patients were The cerebrospinal fluid High-dose for arseno-resistant the effectivity with arseno- treated with (CSF) white blood cell nifurtimox seems Trypanosoma brucei of high dose resistant high-dose (WBC) count decreased more effective than gambiense sleeping nifurtimox for Trypanosoma nifurtimox (30 in all patients except one the previously used sickness: an open trial arsena-resistant brucei gambiense mg/kg/d) for (mean CSF WBC count regimen (15 Title, Author, Journal Objectives of Design Participants Interventions Outcome Results Conclusi,on/ (year, volpp) the study parameters/En Recommendations dpoints in central Zaire T. b. sleeping sickness 30 days. before nifurtimox: mg/kg/d for 60 d), (Pepin, J. et al.) Trans gambiense 117/mm3; after but at the expense R Soc Trop Med Hyg, nifurtimox: 25/mm3), and of significant 1992, Vol. 86, No. 3, trypanosomes toxicity. pp. 254-256 disappeared from the CSF of all 9 patients in whom parasites had been demonstrated before nifurtimox. Among 25 patients seen at least once after treatment, 9 (36%) have relapsed so far. High-dose nifurtimox was significantly toxic: one patient died during treatment and 8 others developed adverse neurological effects. Advances in sleeping To review the Both new substances The current sickness therapy efficacy and constitute effective novel availability of (Van Nieuwenhove, S.) adverse effects therapeutic agents for several effective Ann Soc Belg Med of nifurtimox gambiense sleeping late-stage drugs Trop, 1992, Vol. 72, and DFMO in sickness, including (melarsoprol, Suppl 1, pp. 39-51 the treatment melarsoprol-refractory nifurtimox and of sleeping disease. DFMO is not DFMO), that show sickness very active in rhodesiense synergistic activity sleeping sickness and in experimental experience with models, should nifurtimox in this form of allow the trypanosomiasis is too establishment of limited to draw valid optimum conclusions. The toxicity combination of nifurtimox and DFMO treatment regimens. is not negligible.

Bouteille, B., O. Oukem, et al. (2003). "Treatment perspectives for human ." Fundam Clin Pharmacol 17(2): 171-81. Human African trypanosomiasis (HAT), or sleeping sickness, is currently on the rise. HAT develops in two stages, the first involving the hemolymphatic system, and the second, the neurological system. Left untreated, HAT is invariably fatal. There have been no therapeutic advances in more than 40 years. Stage 1 can be treated with and suramin, but stage 2 can only be treated with melarsoprol, a toxic derivative that has a 2-12% incidence of fatal side-effects (encephalopathy). Eflornithine has never achieved widespread use because it is difficult to administer under field conditions. Nifurtimox has been used successfully in the treatment of American trypanosomiasis, or , but only in small studies or as a compassionate use treatment. There is little research and development for new drugs in this area: only one prodrug is in the clinical development phase, a pentamidine analog that offers hope for the replacement of injectable pentamidine with an orally administered drug. Current efforts appear to be focused on reevaluating older drugs. A course of treatment with melarsoprol for 10 days at 2.2 mg/kg/day is now in the multicenter evaluation phase. Orally administered eflornithine is also slated for reevaluation. In addition, studies of drug combinations are recommended to determine possible combined or synergistic effects and find ways to reduce toxicity.

Burchmore, R. J., P. O. Ogbunude, et al. (2002). "Chemotherapy of human African trypanosomiasis." Curr Pharm Des 8(4): 256-67. Human African trypanosomiasis or sleeping sickness is resurgent [1,2]. The disease is caused by subspecies of the parasitic haemoflagellate, Trypanosoma brucei. Infection starts with the bite of an infected tsetse fly (Glossina spp.). Parasites move from the site of infection to the draining lymphatic vessels and blood stream. The parasites proliferate within the bloodstream and later invade other tissues including the central nervous system. Once they have established themselves within the CNS, a progressive breakdown of neurological function accompanies the disease. Coma precedes death during this late phase. Two forms of the disease are recognised, one caused by Trypanosoma brucei rhodesiense, endemic in Eastern and Southern Africa, in which parasites rapidly invade the CNS causing death within weeks if untreated. T. b. gambiense, originally described in West Africa, but also widespread in Central Africa, proliferates more slowly and can take several years before establishing a CNS- involved infection. Many countries are in the midst of epidemics caused by gambiense- type parasites. Four drugs have been licensed to treat the disease [3]; two of them, pentamidine and suramin, are used prior to CNS involvement. The arsenic-based drug, melarsoprol is used once parasites are established in the CNS. The fourth, eflornithine, is effective against late stage disease caused by T. b. gambiense, but is ineffective against T. b. rhodesiense. Another drug, nifurtimox is licensed for South American trypanosomiasis but also been used in trials against melarsoprol-refractory late sage disease. This review focuses on what is known about modes of action of current drugs and discusses targets for future drug development.

Croft, S. L. (2008). "Kinetoplastida: new therapeutic strategies." Parasite 15(3): 522-7. New formulations and therapeutic switching of the established drugs, and , together with the discovery of , have significantly improved the opportunities for treatment of visceral (VL) chemotherapy. However, for human African trypanosomiasis (HAT), Chagas disease and cutaneous leishmaniases there has been limited progress. For HAT, a novel diamidine, parfuramidine, is in phase III clinical trial for early-stage disease, but for the treatment of late-stage disease there are no new drugs and combinations of eflornithine with melarsoprol or nifurtimox have been the focus of clinical studies. For Chagas disease, different classes of compounds that have validated biochemical targets, sterol biosynthesis methylases and cysteine proteases, are in various stages of development. The genome sequences that are now available for the pathogens that cause the leishmaniases and trypanosomiases, and new methods for rapid validation of targets, are part of the solution to discover new drugs. The integration of medicinal chemistry, , project planning and interaction with the pharma/biotech sector are essential if progress is to be made. Although there are financial constraints, the appearance of new funding sources and not-for-profit product development partnerships offers hope for drug development.

Harder, A., G. Greif, et al. (2001). "Chemotherapeutic approaches to protozoa: kinetoplastida- -current level of knowledge and outlook." Parasitol Res 87(9): 778-80. The possibilities for treating haemoflagellate infections (African trypanosomiasis) are very limited (Table 1; Mehlhorn and Schrevel 1995; Croft 1997; Hunter 1997; Wang 1997; Trouiller and Olliaro 1998). All the available drugs have severe side-effects in humans and animals. Vaccination is not really an option, in view of the wide antigen variability. At present, there are several drug combinations in clinical trials: suramin/eflornithine, suramin/, suramin/pentamidine, melarsoprol/pentamidine, melarsoprol/nifurtimox and nifurtimox/eflornithine. Some of these combinations were successful in treating resistant Trypanosoma brucei rhodesiense and/or T. b. gambiense infections (Keiser et al. 2001). In leishmaniasis, the tendency is still to resort to the old antimony compounds, with their severe side effects. At present, miltefosine is in clinical phase and is the first oral drug against visceral leishmaniasis (Jha et al. 1999). Two drugs are currently used against Chagas' disease, although these do not cure chronic effects. There is no prospect of novel drugs in this indication either (Pecoul et al. 1999; Morel 2000).

Kennedy, P. G. (2008). "The continuing problem of human African trypanosomiasis (sleeping sickness)." Ann Neurol 64(2): 116-26. Human African trypanosomiasis, also known as sleeping sickness, is a neglected disease, and it continues to pose a major threat to 60 million people in 36 countries in sub-Saharan Africa. Transmitted by the bite of the tsetse fly, the disease is caused by protozoan parasites of the genus Trypanosoma and comes in two types: East African human African trypanosomiasis caused by Trypanosoma brucei rhodesiense and the West African form caused by Trypanosoma brucei gambiense. There is an early or hemolymphatic stage and a late or encephalitic stage, when the parasites cross the blood-brain barrier to invade the central nervous system. Two critical current issues are disease staging and drug therapy, especially for late-stage disease. Lumbar puncture to analyze cerebrospinal fluid will remain the only method of disease staging until reliable noninvasive methods are developed, but there is no widespread consensus as to what exactly defines biologically central nervous system disease or what specific cerebrospinal fluid findings should justify drug therapy for late-stage involvement. All four main drugs used for human African trypanosomiasis are toxic, and melarsoprol, the only drug that is effective for both types of central nervous system disease, is so toxic that it kills 5% of patients who receive it. Eflornithine, alone or combined with nifurtimox, is being used increasingly as first-line therapy for gambiense disease. There is a pressing need for an effective, safe oral drug for both stages of the disease, but this will require a significant increase in investment for new drug discovery from Western governments and the pharmaceutical industry.

Legros, D., G. Ollivier, et al. (2002). "Treatment of human African trypanosomiasis--present situation and needs for research and development." Lancet Infect Dis 2(7): 437-40. Human African trypanosomiasis re-emerged in the 1980s. However, little progress has been made in the treatment of this disease over the past decades. The first-line treatment for second-stage cases is melarsoprol, a toxic drug in use since 1949. High therapeutic failure rates have been reported recently in several foci. The alternative, eflornithine, is better tolerated but difficult to administer. A third drug, nifurtimox, is a cheap, orally administered drug not yet fully validated for use in human African trypanosomiasis. No new drugs for second-stage cases are expected in the near future. Because of resistance to and limited number of current treatments, there may soon be no effective drugs available to treat trypanosomiasis patients, especially second-stage cases. Additional research and development efforts must be made for the development of new compounds, including: testing combinations of current trypanocidal drugs, completing the clinical development of nifurtimox and registering it for trypanosomiasis, completing the clinical development of an oral form of eflornithine, pursuing the development of DB 289 and its derivatives, and advancing the pre-clinical development of megazol, eventually engaging firmly in its clinical development. Partners from the public and private sector are already engaged in joint initiatives to maintain the production of current drugs. This network should go further and be responsible for assigning selected teams to urgently needed research projects with funds provided by industry and governments. At the same time, on a long term basis, ambitious research programmes for new compounds must be supported to ensure the sustainable development of new drugs.

Pepin, J. and F. Milord (1994). "The treatment of human African trypanosomiasis." Adv Parasitol 33: 1-47.

Van Voorhis, W. C. (1990). "Therapy and prophylaxis of systemic protozoan infections." Drugs 40(2): 176-202. This article summarises current therapy and prophylaxis for Pneumocystis carinii, Toxoplasma gondii, Leishmania species, African trypanosomes (Trypanosoma brucei gambiense and T. b. rhodesiense), and American trypanosome () infections. Each agent and the disease it causes is briefly reviewed, and current data on the structure, mode of action, indications for treatment, dosage, administration, duration of therapy, efficacy, toxicity, and necessary monitoring during therapy are discussed for each drug. Drugs considered include cotrimoxazole (trimethoprim + sulfamethoxazole), pentamidine, dapsone (diaphenylsulfone), trimetrexate, eflornithine (DFMO), and /clindamycin and pyrimethamine/sulphonamide combinations for Pneumocystis pneumonia; pyrimethamine/sulfadiazine, spiramycin, and clindamycin for toxoplasmosis; pentavalent antimonials ('Pentostam' and 'Glucantime'), pentamidine, amphotericin B, allopurinol, , and itraconazole for leishmaniasis; suramin, pentamidine, melarsoprol, tryparsamide, Mel W, berenil, and eflornithine (DFMO) for African trypanosomiasis; and nifurtimox, and gentian violet for American trypanosomiasis.