Compounds containing 2-substituted imidazole ring for treatment against human African trypanosomiasis

A series of compounds containing 2-substituted imidazoles has been synthesized from imidazole and tested for its biological activity against human African trypanosomiasis (HAT). The 2-substituted 5-nitroimidazoles such as fexinidazole (7a) and 1-[4-(1-methyl-5-nitro-1H-imidazol-2-ylmethoxy)-pyridin-2-yl-piperazine (9e) exhibited potent activity against T. brucei in vitro with low cytotoxicity and good solubility. The presence of the NO₂ group at the 5-position of the imidazole ring in 2-substituted imidazoles is the crucial factor to inhibit T. brucei.     

Entomological survey in the historical focus of human African trypanosomiasis of Bendje (Gabon)

The situation of human African trypanosomiasis (sleeping sickness) is poorly known in Gabon. Most of the historical foci have not been investigated for more than 15 years. Few cases are passively recorded from the historical focus of Bendjé; they involved mainly fishermen but determining their contamination site is difficult because of their mobility due to their activity. The presence of these cases in that focus could favour its reactivation if the vector is still there. In order to assess a potential transmission risk in that area, an entomological survey has been carried out in it. Traps were set up during four days in different habitats used by humans during their daily activities. Three species of tsetse flies (Glossina palpalis palpalis, G. pallicera newsteadi and G. caliginea) were caught and two species of trypanosomes (Trypanosoma vivax and T. brucei s.l.) were identified by PCR. These results suggest the presence of an animal transmission cycle. Human-flies contact was confirmed in all type of habitats but no transmission was quantified in the mangrove.     

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.     

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.     

In vitro induction of microglial and endothelial cell apoptosis by cerebrospinal fluids from patients with human African trypanosomiasis

In human African trypanosomiasis, trypanosomes first develop in the blood and lymph (Stage 1), then spread to the central nervous system (CNS) (Stage 2). Disruption of the blood-brain barrier of unknown mechanism occurs in Stage 2 disease. The hypothesis that cerebrospinal fluids (CSF) from African trypanosomiasis patients might contain factor(s) able to induce apoptosis in endothelial cells led us to evaluate this effect by two methods, the TdT-mediated dUTP nick end labelling (TUNEL) method and the measurement of soluble nucleosomes released by apoptotic cells in culture supernatant by ELISA. Apoptosis induction by CSF was also studied with microglial cells, the resident macrophages in the brain, which participate in the blood-brain barrier in the perivascular area. In contrast with control CSF, African trypanosomiasis patients' CSF induced apoptosis in both microglial and endothelial cells. The results obtained with the two methods correlated well, and showed that Stage 2 CSF induced apoptosis at higher levels in microglial cells, whereas the disease stage was not decisive for apoptosis induction in endothelial cells. We measured soluble Fas ligand (sFasL) and anti-Fas antibodies levels, two potent inducers of the Fas signalling pathway leading to apoptosis, in CSF from African trypanosomiasis patients and controls. CSF from African trypanosomiasis patients contained sFasL, and anti-Fas antibodies at higher levels than in controls. Stage 2 CSF contained more sFasL than Stage 1 CSF, and anti-Fas antibodies were detected only in Stage 2 CSF. Caspase-8 inhibitor effect and statistical data suggest that other pro-apoptotic factors may be involved in some CSF-induced apoptosis. Apoptosis induction may participate in the pathogenesis during African trypanosomiasis, and the presence of sFasL and anti-Fas antibodies may provide new tools for diagnosis and prognosis of the disease.     

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.     

Assessment of a pretomanid analogue library for African trypanosomiasis: Hit-to-lead studies on 6-substituted 2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]thiazine 8-oxides

A 900 compound nitroimidazole-based library derived from our pretomanid backup program with TB Alliance was screened for utility against human African trypanosomiasis (HAT) by the Drugs for Neglected Diseases initiative. Potent hits included 2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]thiazine 8-oxides, which surprisingly displayed good metabolic stability and excellent cell permeability. Following comprehensive mouse pharmacokinetic assessments on four hits and determination of the most active chiral form, a thiazine oxide counterpart of pretomanid (24) was identified as the best lead. With once daily oral dosing, this compound delivered complete cures in an acute infection mouse model of HAT and increased survival times in a stage 2 model, implying the need for more prolonged CNS exposure. In preliminary SAR findings, antitrypanosomal activity was reduced by removal of the benzylic methylene but enhanced through a phenylpyridine-based side chain, providing important direction for future studies.     

Trypanosoma brucei: Immunisation with plasmid DNA encoding invariant surface glycoprotein gene is able to induce partial protection in experimental African trypanosomiasis

Trypanosoma brucei is the etiological agent responsible for African trypanosomiasis, an infectious pathology which represents a serious problem of public health and economic losses in Sub-Saharan Africa. As one of the foremost neglected illnesses, few resources have been available for the development of vaccines or new drugs, in spite of the current therapeutical drugs showing little efficiency and high toxicity. Hence, it is obviously important to widen effective therapeutics and preventive strategies against African trypanosomiasis. In this work, we use the DNA vaccine model to evaluate immunisation effectiveness in mice challenged with Trypanosoma brucei brucei. We demonstrate that Balb/C mice immunised intramuscularly with a single dose of a DNA plasmid encoding a bloodstream-stage specific invariant surface glycoprotein (ISG) are partially protected from a lethal dose of T. b. brucei. Interestingly, the surviving animals show high levels of IgG2a anti-trypanosoma antibodies, suggesting that the Th1 response profile seems important for the induced mechanisms of immune protection.     

Glutathione reductase (EC 1.6.4.2.) in experimental trypanosomiasis

The activity of glutathione reductase (GHSR) in extracts of kidney, liver and testis of rats infected with Trypanosoma congolense decreased with every wave of parasitemia. The implications of these observations as they relate to the risk of oxidative stress are discussed.     

A new transmission risk index for human African trypanosomiasis and its application in the identification of sites of high transmission of sleeping sickness in the Fontem focus of southwest Cameroon

A new index for the risk for transmission of human African trypanosomiasis was developed from an earlier index by adding terms for the proportion of tsetse infected with Trypanosoma brucei gambiense group 1 and the contribution of animals to tsetse diet. The validity of the new index was then assessed in the Fontem focus of southwest Cameroon. Averages of 0.66 and 4.85 Glossina palpalis palpalis (Diptera: Glossinidae) were caught per trap/day at the end of one rainy season (November) and the start of the next (April), respectively. Of 1596 tsetse flies examined, 4.7% were positive for Trypanosoma brucei s.l. midgut infections and 0.6% for T. b. gambiense group 1. Among 184 bloodmeals identified, 55.1% were from pigs, 25.2% from humans, 17.6% from wild animals and 1.2% from goats. Of the meals taken from humans, 81.5% were taken at sites distant from pigsties. At the end of the rainy season, catches were low and similar between biotopes distant from and close to pigsties, but the risk for transmission was greatest at sites distant from the sties, suggesting that the presence of pigs reduced the risk to humans. At the beginning of the rainy season, catches of tsetse and risk for transmission were greatest close to the sties. In all seasons, there was a strong correlation between the old and new indices, suggesting that both can be used to estimate the level of transmission, but as the new index is the more comprehensive, it may be more accurate.     

African trypanosomes and brain infection – the unsolved question

African trypanosomes induce sleeping sickness. The parasites are transmitted during the blood meal of a tsetse fly and appear primarily in blood and lymph vessels, before they enter the central nervous system. During the latter stage, trypanosomes induce a deregulation of sleep–wake cycles and some additional neurological disorders. Historically, it was assumed that trypanosomes cross the blood–brain barrier and settle somewhere between the brain cells. The brain, however, is a strictly controlled and immune‐privileged area that is completely surrounded by a dense barrier that covers the blood vessels: this is the blood–brain barrier. It is known that some immune cells are able to cross this barrier, but this requires a sophisticated mechanism and highly specific cell–cell interactions that have not been observed for trypanosomes within the mammalian host. Interestingly, trypanosomes injected directly into the brain parenchyma did not induce an infection. Likewise, after an intraperitoneal infection of rats, Trypanosoma brucei brucei was not observed within the brain, but appeared readily within the cerebrospinal fluid (CSF) and the meninges. Therefore, the parasite did not cross the blood–brain barrier, but the blood–CSF barrier, which is formed by the choroid plexus, i.e. the part of the ventricles where CSF is produced from blood. While there is no question that trypanosomes are able to invade the brain to induce a deadly encephalopathy, controversy exists about the pathway involved. This review lists experimental results that support crossing of the blood–brain barrier and of the blood–CSF barrier and discuss the implications that either pathway would have on infection progress and on the survival strategy of the parasite. For reasons discussed below, we prefer the latter pathway and suggest the existence of an additional distinct meningeal stage, from which trypanosomes could invade the brain via the Virchow–Robin space thereby bypassing the blood–brain barrier. We also consider healthy carriers, i.e. people living symptomless with the disease for up to several decades, and discuss implications the proposed meningeal stage would have for new anti‐trypanosomal drug development. Considering the re‐infection of blood, a process called relapse, we discuss the likely involvement of the newly described glymphatic connection between the meningeal space and the lymphatic system, that seems also be important for other infectious diseases.     

Discovery of halo-nitrobenzamides with potential application against human African trypanosomiasis

A series of halo-nitrobenzamide were synthesized and evaluated for their ability to block proliferation of Trypanosoma brucei brucei. A number of these compounds had significant activity against the parasite, particularly 2-chloro-N-(4-chlorophenyl)-5-nitrobenzamide 17 which exhibited low micromolar inhibitory potency against T. brucei and selectivity towards both malaria and mammalian cells.     

Novel naphthoquinone derivatives: Synthesis and activity against human African trypanosomiasis

A series of naphthoquinone derivatives has been synthesized and tested for its biological activity against human African trypanosomiasis. The use of reverse micellar medium not only enhanced the conversion rate, but also showed selectivity towards mono-coupled product in aryl chloride–aniline coupling reactions. Two derivatives of naphthoquinone (9b and 9c) exhibited potent activity against Trypanosoma brucei in vitro with low cytotoxicity.     

Synthesis and antitrypanosomal evaluation of derivatives of N-benzyl-1,2-dihydroquinolin-6-ols: Effect of core substitutions and salt formation

Analogs of the trypanocidal lead compound 1-benzyl-1,2-dihydro-2,2,4-trimethylquinolin-6-yl acetate were prepared to extend the structure-activity relationship in this series of molecules, improve the in vivo antitrypanosomal activity of the lead, and determine whether ester prodrugs are needed to overcome the instability of the dihydroquinolin-6-ols. Two of the most active compounds identified in this study were 1-benzyl-1,2-dihydro-2,2,4-trimethylquinolin-6-ol hydrochloride and 1-(2-methoxy)benzyl-1,2-dihydro-2,2,4-trimethylquinolin-6-ol hydrochloride. These stable solids possessed low nanomolar IC₅₀ values against Trypanosoma brucei rhodesiense STIB900 in vitro and provided cures in an early treatment acute mouse model of African trypanosomiasis when given ip at 50 mg/kg/day for four consecutive days.