Combination chemotherapy with a substance P receptor antagonist (aprepitant) and melarsoprol in a mouse model of human African trypanosomiasis

Drug therapy for late-stage (encephalitic) human African trypanosomiasis (HAT) is currently very unsatisfactory with the most commonly used drug, melarsoprol, having a 5% overall mortality. There is evidence in a mouse model of HAT that Substance P (SP) receptor antagonism reduces the neuroinflammatory reaction to CNS trypanosome infection. In this study we investigated the effects of combination chemotherapy with melarsoprol and a humanised SP receptor antagonist aprepitant (EMEND) in this mouse model. The melarsoprol/aprepitant drug combination did not produce any clinical signs of illness in mice with CNS trypanosome infection. This lack of any additional or unexpected CNS toxicity in the mouse model of CNS HAT provides valuable safety data for the future possible use of this drug combination in patients with late-stage HAT.     

Melarsoprol cyclodextrin inclusion complexes as promising oral candidates for the treatment of human African trypanosomiasis

Human African trypanosomiasis (HAT), or sleeping sickness, results from infection with the protozoan parasites Trypanosoma brucei (T. b.) gambiense or T. b. rhodesiense and is invariably fatal if untreated. There are 60 million people at risk from the disease throughout sub-Saharan Africa. The infection progresses from the haemolymphatic stage where parasites invade the blood, lymphatics and peripheral organs, to the late encephalitic stage where they enter the central nervous system (CNS) to cause serious neurological disease. The trivalent arsenical drug melarsoprol (Arsobal) is the only currently available treatment for CNS-stage T. b. rhodesiense infection. However, it must be administered intravenously due to the presence of propylene glycol solvent and is associated with numerous adverse reactions. A severe post-treatment reactive encephalopathy occurs in about 10% of treated patients, half of whom die. Thus melarsoprol kills 5% of all patients receiving it. Cyclodextrins have been used to improve the solubility and reduce the toxicity of a wide variety of drugs. We therefore investigated two melarsoprol cyclodextrin inclusion complexes; melarsoprol hydroxypropyl-β-cyclodextrin and melarsoprol randomly-methylated-β-cyclodextrin. We found that these compounds retain trypanocidal properties in vitro and cure CNS-stage murine infections when delivered orally, once per day for 7-days, at a dosage of 0.05 mmol/kg. No overt signs of toxicity were detected. Parasite load within the brain was rapidly reduced following treatment onset and magnetic resonance imaging showed restoration of normal blood-brain barrier integrity on completion of chemotherapy. These findings strongly suggest that complexed melarsoprol could be employed as an oral treatment for CNS-stage HAT, delivering considerable improvements over current parenteral chemotherapy.     

Update on field use of the available drugs for the chemotherapy of human African trypanosomiasis

Despite the fact that eflornithine was considered as the safer drug to treat human African trypanosomiasis (HAT) and has been freely available since 2001, the difficulties in logistics and cost burden associated with this drug meant that the toxic melarsoprol remained the drug of choice. The World Health Organization responded to the situation by designing a medical kit containing all the materials needed to use eflornithine, and by implementing a training and drugs distribution programme which has allowed a transition to this much safer treatment. The introduction of the combination of nifurtimox and eflornithine (NECT) has accelerated the shift from melarsoprol to the best treatment available, due to reduced dosage and treatment time for eflornithine that has significantly lessened the cost and improved the burden of logistics encountered during treatment and distribution. The decrease in the use of more dangerous but cheaper melarsoprol has meant a rise in the per patient cost of treating HAT. Although NECT is cheaper than eflornithine monotherapy, an unexpected consequence has been a continuing rise in the per patient cost of treating HAT. The ethical decision of shifting to the best available treatment imposes a financial burden on HAT control programmes that might render long-term application unsustainable. These factors call for continuing research to provide new safer and more effective drugs that are simple to administer and cheaper when compared to current drugs.     

Human African trypanosomiasis: potential therapeutic benefits of an alternative suramin and melarsoprol regimen

Treatment of late-stage human African trypanosomiasis is complicated by the presence of trypanosomes within the central nervous system (CNS). The regimen commonly prescribed to treat CNS-stage disease involves the use of the trypanocidal drugs suramin and melarsoprol. Suramin does not cross the blood–brain barrier efficiently and therefore, at normal dosages, will not cure CNS-stage infections. An initial treatment with suramin is given to eliminate the parasites from the peripheral tissues. This is followed by a course of intravenous melarsoprol, which can enter the CNS. However, melarsoprol not only produces severe adverse reactions but also is extremely painful to administer. One possible method to help alleviate these problems is to reduce the total amount of melarsoprol in the treatment regimen. This study indicates a synergism between suramin and melarsoprol and demonstrates that experimental murine CNS-trypanosomiasis can be cured with a single intraperitoneal dose of 20 mg/kg suramin followed almost immediately by 0.05 ml (4.5 μmol) topical melarsoprol. These dosages will not cure the infection when administered as monotherapies. Moreover, the timing of the drug administration appears to be crucial to the successful outcome of the regimen. If the interval between injection of suramin and application of topical melarsoprol is extended from 15 min to 3 or 7 days, the infections are not cured. Although extended relapse times occur following these regimens when compared with monotherapy approaches. Thus, there is strong evidence that injected suramin and topical melarsoprol should be given almost simultaneously to achieve the most effective combination of the two drugs.     

Kinetoplastida: new therapeutic strategies

New formulations and therapeutic switching of the established drugs, amphotericin B and paromomycin, together with the discovery of miltefosine, have significantly improved the opportunities for treatment of visceral leishmaniasis (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, pharmacokinetics, 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.     

Experimental therapy of African trypanosomiasis with a nanobody-conjugated human trypanolytic factor

High systemic drug toxicity and increasing prevalence of drug resistance hampers efficient treatment of human African trypanosomiasis (HAT). Hence, development of new highly specific trypanocidal drugs is necessary. Normal human serum (NHS) contains apolipoprotein L-I (apoL-I), which lyses African trypanosomes except resistant forms such as Trypanosoma brucei rhodesiense. T. b. rhodesiense expresses the apoL-I-neutralizing serum resistance-associated (SRA) protein, endowing this parasite with the ability to infect humans and cause HAT. A truncated apoL-I (Tr-apoL-I) has been engineered by deleting its SRA-interacting domain, which makes it lytic for T. b. rhodesiense. Here, we conjugated Tr-apoL-I with a single-domain antibody (nanobody) that efficiently targets conserved cryptic epitopes of the variant surface glycoprotein (VSG) of trypanosomes to generate a new manmade type of immunotoxin with potential for trypanosomiasis therapy. Treatment with this engineered conjugate resulted in clear curative and alleviating effects on acute and chronic infections of mice with both NHS-resistant and NHS-sensitive trypanosomes.     

Melarsoprol- and pentamidine-resistant Trypanosoma brucei rhodesiense populations and their cross-resistance

Resistance to melarsoprol and pentamidine was induced in bloodstream-form Trypanosoma brucei rhodesiense STIB 900 in vitro, and drug sensitivity was determined for melarsoprol, pentamidine and furamidine. The resistant populations were also inoculated into immunosuppressed mice to verify infectivity and to monitor whether rodent passage selects for clones with altered drug sensitivity. After proliferation in the mouse, trypanosomes were isolated and their IC(50) values to the three drugs were determined. To assess the stability of drug-induced resistance, drug pressure was ceased for 2 months and the drug sensitivity was determined again. Resistance was stable, with a few exceptions that are discussed. Drug IC(50)s indicated cross-resistance among all drugs, but to varying extents: resistance of the melarsoprol-selected and pentamidine-selected trypanosomes to pentamidine was the same, but the pentamidine-selected trypanosome population showed lower resistance to melarsoprol than the melarsoprol-selected trypanosomes. Interestingly, both resistant populations revealed the same intermediate cross-resistance to furamidine. Resistant trypanosome populations were characterised by molecular means, referring to the status of the TbAT1 gene. The melarsoprol-selected population apparently had lost TbAT1, whereas in the pentamidine-selected trypanosome population it was still present.     

Chemotherapy against human African trypanosomiasis: is there a road to success?

For over fifty years, human African trypanosomiasis (HAT, sleeping sickness) has been treated with suramin, pentamidine and the very toxic organo-arsenical melarsoprol that was the only drug available for effective treatment of the second stage of the disease. Recently there have been significant efforts using molecular and biochemical approaches to drug design, including high-throughput screening, but the number of lead compounds with promising activity against T. brucei spp. and an acceptable toxicity index has remained astonishingly small. Clinical research continues to be difficult due to the economic constraints and the complexity of trials on a low prevalence disease in remote and impoverished African regions. Despite those limitations the situation for the patients is improving thanks to the combination of a number of critical factors. By the late 1990s the disease had reached epidemic levels that triggered political support. WHO would sign a donation agreement with the manufacturers for all drugs to treat HAT. A result of this agreement was that eflornithine which is much safer than melarsoprol became available and widely used by non-governmental organizations. The Impamel I and II programmes demonstrated that against all odds the conduct of clinical trials on HAT was feasible. This allowed the initiation of trials on combination therapies which eventually resulted in the nifurtimox-eflornithine combination treatment (NECT). This combination is currently being introduced as first line treatment, and there is even the prospect of having a new compound, fexinidazole, in the development pipeline. This review summarizes the key information about the existing drugs and gives a comprehensive summary about the recent and currently ongoing efforts towards new drugs.     

A multiplex PCR that discriminates between Trypanosoma brucei brucei and zoonotic T. b. rhodesiense

Two subspecies of Trypanosoma brucei s.l. co-exist within the animal populations of Eastern Africa; T. b. brucei a parasite which only infects livestock and wildlife and T. b. rhodesiense a zoonotic parasite which infects domestic livestock, wildlife, and which in humans, results in the disease known as Human African Trypanosomiasis (HAT) or sleeping sickness. In order to assess the risk posed to humans from HAT it is necessary to identify animals harbouring potentially human infective parasites. The multiplex PCR method described here permits differentiation of human and non-human infective parasites T. b. rhodesiense and T. b. brucei based on the presence or absence of the SRA gene (specific for East African T. b. rhodesiense), inclusion of GPI-PLC as an internal control indicates whether sufficient genomic material is present for detection of a single copy T. brucei gene in the PCR reaction.     

Genotypic Status of the TbAT1/P2 Adenosine Transporter of Trypanosoma brucei gambiense Isolates from Northwestern Uganda following Melarsoprol Withdrawal

Background

The development of arsenical and diamidine resistance in Trypanosoma brucei is associated with loss of drug uptake by the P2 purine transporter as a result of alterations in the corresponding T. brucei adenosine transporter 1 gene (TbAT1). Previously, specific TbAT1 mutant type alleles linked to melarsoprol treatment failure were significantly more prevalent in T. b. gambiense from relapse patients at Omugo health centre in Arua district. Relapse rates of up to 30% prompted a shift from melarsoprol to eflornithine (α-difluoromethylornithine, DFMO) as first-line treatment at this centre. The aim of this study was to determine the status of TbAT1 in recent isolates collected from T. b. gambiense sleeping sickness patients from Arua and Moyo districts in Northwestern Uganda after this shift in first-line drug choice.

Methodology and results

Blood and cerebrospinal fluids of consenting patients were collected for DNA preparation and subsequent amplification. All of the 105 isolates from Omugo that we successfully analysed by PCR-RFLP possessed the TbAT1 wild type allele. In addition, PCR/RFLP analysis was performed for 74 samples from Moyo, where melarsoprol is still the first line drug; 61 samples displayed the wild genotype while six were mutant and seven had a mixed pattern of both mutant and wild-type TbAT1. The melarsoprol treatment failure rate at Moyo over the same period was nine out of 101 stage II cases that were followed up at least once. Five of the relapse cases harboured mutant TbAT1, one had the wild type, while no amplification was achieved from the remaining three samples.

Conclusions/significance

The apparent disappearance of mutant alleles at Omugo may correlate with melarsoprol withdrawal as first-line treatment. Our results suggest that melarsoprol could successfully be reintroduced following a time lag subsequent to its replacement. A field-applicable test to predict melarsoprol treatment outcome and identify patients for whom the drug can still be beneficial is clearly required. This will facilitate cost-effective management of HAT in rural resource-poor settings, given that eflornithine has a much higher logistical requirement for its application.     

The antitrypanosomal drug melarsoprol competitively inhibits thiamin uptake in mouse neuroblastoma cells

Melarsoprol is the main drug used for the treatment of late-stage sleeping sickness, although it causes severe side-effects such as encephalopathy and polyneuropathy leading to death in some patients. Recent data suggest that melarsoprol and its active metabolite melarsenoxide interfere with thiamin transport and metabolism in E. coli and yeast, but there are no data concerning their possible effects on thiamin metabolism in mammalian cells. We tested both drugs on thiamin transport in cultured mouse neuroblastoma cells using ¹⁴C-labeled thiamin. Melarsoprol, competitively inhibits high-affinity thiamin transport in mouse neuroblastoma cells with a K_i of 44 μmol/L. However, the active compound melarsenoxide has no inhibitory effect. This suggests that the side effects of melarsoprol treatment are unlikely to be due to inhibition of thiamin transport by melarsenoxide, its main metabolite in the brain.     

Clinical presentation of T.b. rhodesiense sleeping sickness in second stage patients from Tanzania and Uganda

Background

A wide spectrum of disease severity has been described for Human African Trypanosomiasis (HAT) due to Trypanosoma brucei rhodesiense (T.b. rhodesiense), ranging from chronic disease patterns in southern countries of East Africa to an increase in virulence towards the north. However, only limited data on the clinical presentation of T.b. rhodesiense HAT is available. From 2006-2009 we conducted the first clinical trial program (Impamel III) in T.b. rhodesiense endemic areas of Tanzania and Uganda in accordance with international standards (ICH-GCP). The primary and secondary outcome measures were safety and efficacy of an abridged melarsoprol schedule for treatment of second stage disease. Based on diagnostic findings and clinical examinations at baseline we describe the clinical presentation of T.b. rhodesiense HAT in second stage patients from two distinct geographical settings in East Africa.

Methodology/Principal Findings:

138 second stage patients from Tanzania and Uganda were enrolled. Blood samples were collected for diagnosis and molecular identification of the infective trypanosomes, and T.b. rhodesiense infection was confirmed in all trial subjects. Significant differences in diagnostic parameters and clinical signs and symptoms were observed: the median white blood cell (WBC) count in the cerebrospinal fluid (CSF) was significantly higher in Tanzania (134cells/mm³) than in Uganda (20cells/mm³; p<0.0001). Unspecific signs of infection were more commonly seen in Uganda, whereas neurological signs and symptoms specific for HAT dominated the clinical presentation of the disease in Tanzania. Co-infections with malaria and HIV did not influence the clinical presentation nor treatment outcomes in the Tanzanian study population.

Conclusions/Significance

We describe a different clinical presentation of second stage T.b. rhodesiense HAT in two distinct geographical settings in East Africa. In the ongoing absence of sensitive diagnostic tools and safe drugs to diagnose and treat second stage T.b. rhodesiense HAT an early identification of the disease is essential. A detailed understanding of the clinical presentation of T.b. rhodesiense HAT among health personnel and affected communities is vital, and awareness of regional characteristics, as well as implications of co-infections, can support decision making and differential diagnosis.     

Diagnostic and neuropathogenesis issues in human African trypanosomiasis

Human African trypanosomiasis, also known as sleeping sickness, is caused by protozoan parasites of the genus Trypanosoma, and is a major cause of human mortality and morbidity. The East African and West African variants, caused by Trypanosma brucei rhodesiense and Trypanosoma brucei gambiense, respectively, differ in their presentation but the disease is fatal if untreated. Accurate staging of the disease into the early haemolymphatic stage and the late encephalitic stage is critical as the treatment for the two stages is different. The only effective drug for late stage disease, melarsoprol, which crosses the blood-brain barrier, is followed by a severe post-treatment reactive encephalopathy in 10% of cases of which half die. There is no current consensus on the diagnostic criteria for CNS involvement and the specific indications for melarsoprol therapy also differ. There is a pressing need for a quick, simple, cheap and reliable diagnostic test to diagnose Human African trypanosomiasis in the field and also to determine CNS invasion. Cerebrospinal fluid and plasma analyses in patients with Human African trypanosomiasis have indicated a role for both pro-inflammatory and counter-inflammatory cytokines in determining the severity of the meningoencephalitis of late stage disease, and, at least in T. b. rhodesiense infection, the balance of these opposing cytokines may be critical. Rodent models of Human African trypanosomiasis have proved very useful in modelling the post-treatment reactive encephalopathy of humans and have demonstrated the central role of astrocyte activation and cytokine balances in determining CNS disease. Such animal models have also allowed a greater understanding of the more direct mechanisms of trypanosome infection on CNS function including the disruption of circadian rhythms, as well as the immunological determinants of passage of trypanosomes across the blood-brain barrier.     

Overexpression of the putative thiol conjugate transporter TbMRPA causes melarsoprol resistance in Trypanosoma brucei

Melaminophenyl arsenical drugs are a mainstay of chemotherapy against late-stage African sleeping sickness, but drug resistance is increasingly prevalent. We describe here the characterization of two genes encoding putative metal-thiol conjugate transporters from Trypanosoma brucei. The two proteins, TbMRPA and TbMRPE, were each overexpressed in trypanosomes, with or without co-expression of two key enzymes in trypanothione biosynthesis, ornithine decarboxylase and gamma-glutamyl-cysteine synthetase. Overexpression of gamma-glutamyl-cysteine synthetase resulted in a twofold increase in cellular trypanothione, whereas overexpression of ornithine decarboxylase had no effect on the trypanothione level. The overexpression of TbMRPA resulted in a 10-fold increase in the IC50 of melarsoprol. The overexpression of the trypanothione biosynthetic enzymes alone gave two- to fourfold melarsoprol resistance, but did not enhance resistance caused by MRPA. Overexpression of TbMRPE had little effect on susceptibility to melarsoprol but did give two- to threefold resistance to suramin.     

Human African trypanosomiasis: connecting parasite and host genetics

In West and Central Africa, the protozoan parasite Trypanosoma brucei (T. b.) gambiense causes a chronic form of Human African trypanosomiasis (HAT) that might last several years, whereas T. b. rhodesiense refers to an acute form in East Africa that lasts weeks to months. Without treatment, both forms can cause death. Diagnosis relies on detecting parasites in blood, lymph or cerebrospinal fluid. HAT was no longer considered a public health problem in the 1960s, but it returned to alarming levels in the 1990s. After intensifying case detection and treatment, WHO recently declared the situation is under control. However, research based on host and trypanosome interactions should be encouraged to help develop innovative tools for HAT diagnosis and treatment to prevent re-emergence.     

Mechanisms of arsenical and diamidine uptake and resistance in Trypanosoma brucei

Sleeping sickness, caused by Trypanosoma brucei spp., has become resurgent in sub-Saharan Africa. Moreover, there is an alarming increase in treatment failures with melarsoprol, the principal agent used against late-stage sleeping sickness. In T. brucei, the uptake of melarsoprol as well as diamidines is thought to be mediated by the P2 aminopurine transporter, and loss of P2 function has been implicated in resistance to these agents. The trypanosomal gene TbAT1 has been found to encode a P2-type transporter when expressed in yeast. Here we investigate the role of TbAT1 in drug uptake and drug resistance in T. brucei by genetic knockout of TbAT1. Tbat1-null trypanosomes were deficient in P2-type adenosine transport and lacked adenosine-sensitive transport of pentamidine and melaminophenyl arsenicals. However, the null mutants were only slightly resistant to melaminophenyl arsenicals and pentamidine, while resistance to other diamidines such as diminazene was more pronounced. Nevertheless, the reduction in drug sensitivity might be of clinical significance, since mice infected with tbat1-null trypanosomes could not be cured with 2 mg of melarsoprol/kg of body weight for four consecutive days, whereas mice infected with the parental line were all cured by using this protocol. Two additional pentamidine transporters, HAPT1 and LAPT1, were still present in the null mutant, and evidence is presented that HAPT1 may be responsible for the residual uptake of melaminophenyl arsenicals. High-level arsenical resistance therefore appears to involve the loss of more than one transporter.     

Isothermal microcalorimetry, a new tool to monitor drug action against Trypanosoma brucei and Plasmodium falciparum

Isothermal microcalorimetry is an established tool to measure heat flow of physical, chemical or biological processes. The metabolism of viable cells produces heat, and if sufficient cells are present, their heat production can be assessed by this method. In this study, we investigated the heat flow of two medically important protozoans, Trypanosoma brucei rhodesiense and Plasmodium falciparum. Heat flow signals obtained for these pathogens allowed us to monitor parasite growth on a real-time basis as the signals correlated with the number of viable cells. To showcase the potential of microcalorimetry for measuring drug action on pathogenic organisms, we tested the method with three antitrypanosomal drugs, melarsoprol, suramin and pentamidine and three antiplasmodial drugs, chloroquine, artemether and dihydroartemisinin, each at two concentrations on the respective parasite. With the real time measurement, inhibition was observed immediately by a reduced heat flow compared to that in untreated control samples. The onset of drug action, the degree of inhibition and the time to death of the parasite culture could conveniently be monitored over several days. Microcalorimetry is a valuable element to be added to the toolbox for drug discovery for protozoal diseases such as human African trypanosomiasis and malaria. The method could probably be adapted to other protozoan parasites, especially those growing extracellularly.     

From Health Advice to Taboo: Community Perspectives on the Treatment of Sleeping Sickness in the Democratic Republic of Congo,a Qualitative Study

Background

Socio-cultural and economic factors constitute real barriers for uptake of screening and treatment of Human African Trypanosomiasis (HAT) in the Democratic Republic of Congo (DRC). Better understanding and addressing these barriers may enhance the effectiveness of HAT control.

Methods

We performed a qualitative study consisting of semi-structured interviews and focus group discussions in the Bandundu and Kasaï Oriental provinces, two provinces lagging behind in the HAT elimination effort. Our study population included current and former HAT patients, as well as healthcare providers and program managers of the national HAT control program. All interviews and discussions were voice recorded on a digital device and data were analysed with the ATLAS.ti software.

Findings

Health workers and community members quoted a number of prohibitions that have to be respected for six months after HAT treatment: no work, no sexual intercourse, no hot food, not walking in the sun. Violating these restrictions is believed to cause serious, and sometimes deadly, complications. These strong prohibitions are well-known by the community and lead some people to avoid HAT screening campaigns, for fear of having to observe such taboos in case of diagnosis.

Discussion

The restrictions originally aimed to mitigate the severe adverse effects of the melarsoprol regimen, but are not evidence-based and became obsolete with the new safer drugs. Correct health information regarding HAT treatment is essential. Health providers should address the perspective of the community in a constant dialogue to keep abreast of unintended transformations of meaning.