An Overview on Target-Based Drug Design against Kinetoplastid Protozoan Infections: Human African Trypanosomiasis, Chagas Disease and Leishmaniases.

The protozoan diseases Human African Trypanosomiasis (HAT), Chagas disease (CD), and leishmaniases span worldwide and therefore their impact is a universal concern. The present regimen against kinetoplastid protozoan infections is poor and insufficient. Target-based design expands the horizon of drug design and development and offers novel chemical entities and potential drug candidates to the therapeutic arsenal against the aforementioned neglected diseases. In this review, we report the most promising targets of the main kinetoplastid parasites, as well as their corresponding inhibitors. This overview is part of the Special Issue, entitled "Advances of Medicinal Chemistry against Kinetoplastid Protozoa (Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp.) Infections: Drug Design, Synthesis and Pharmacology".

Six New Phenylpropanoid Derivatives from Chemically Converted Extract of Alpinia galanga (L.) and Their Antiparasitic Activities.

Chemical conversion of the extract of natural resources is a very attractive way to expand the chemical space to discover bioactive compounds. In order to search for new medicines to treat parasitic diseases that cause high morbidity and mortality in affected countries in the world, the ethyl acetate extract from the rhizome of Alpinia galanga (L.) has been chemically converted by epoxidation using dioxirane generated in situ. The biological activity of chemically converted extract (CCE) of A. galanga (L.) significantly increased the activity against Leishmania major up to 82.6 ± 6.2 % at 25 µg/mL (whereas 2.7 ± 0.8% for the original extract). By bioassay-guided fractionation, new phenylpropanoids (1-6) and four known compounds, hydroquinone (7), 4-hydroxy(4-hydroxyphenyl)methoxy)benzaldehyde (8), isocoumarin cis 4-hydroxymelein (9), and (2S,3S,6R,7R,9S,10S)-humulene triepoxide (10) were isolated from CCE. The structures of isolated compounds were determined by spectroscopic analyses of 1D and 2D NMR, IR, and MS spectra. The most active compound was hydroquinone (7) with IC50 = 0.37 ± 1.37 µg/mL as a substantial active principle of CCE. In addition, the new phenylpropanoid 2 (IC50 = 27.8 ± 0.34 µg/mL) also showed significant activity against L. major compared to the positive control miltefosine (IC50 = 7.47 ± 0.3 µg/mL). The activities of the isolated compounds were also evaluated against Plasmodium falciparum, Trypanosoma brucei gambisense and Trypanosoma brucei rhodeisense. Interestingly, compound 2 was selectively active against trypanosomes with potent activity. To the best of our knowledge, this is the first report on the bioactive "unnatural" natural products from the crude extract of A. galanga (L.) by chemical conversion and on its activities against causal pathogens of leishmaniasis, trypanosomiasis, and malaria.

Adaptation of Trypanosoma rhodesiense to hypohaptoglobinaemic serum requires transcription of the APOL1 resistance gene in a RNA polymerase I locus.

Human apolipoprotein L1 (APOL1) kills African trypanosomes except Trypanosoma rhodesiense and Trypanosoma gambiense, the parasites causing sleeping sickness. APOL1 uptake into trypanosomes is favoured by its association with the haptoglobin-related protein-haemoglobin complex, which binds to the parasite surface receptor for haptoglobin-haemoglobin. As haptoglobin-haemoglobin can saturate the receptor, APOL1 uptake is increased in haptoglobin-poor (hypohaptoglobinaemic) serum (HyHS). While T. rhodesiense resists APOL1 by RNA polymerase I (pol-I)-mediated expression of the serum resistance-associated (SRA) protein, T. gambiense resists by pol-II-mediated expression of the T. gambiense-specific glycoprotein (TgsGP). Moreover, in T. gambiense resistance to HyHS is linked to haptoglobin-haemoglobin receptor inactivation by mutation. We report that unlike T. gambiense, T. rhodesiense possesses a functional haptoglobin-haemoglobin receptor, and that like T. gambiense experimentally provided with active receptor, this parasite is killed in HyHS because of receptor-mediated APOL1 uptake. However, T. rhodesiense could adapt to low haptoglobin by increasing transcription of SRA. When assayed in Trypanosoma brucei, resistance to HyHS occurred with pol-I-, but not with pol-II-mediated SRA expression. Similarly, T. gambiense provided with active receptor acquired resistance to HyHS only when TgsGP was moved to a pol-I locus. Thus, transcription by pol-I favours adaptive gene regulation, explaining the presence of SRA in a pol-I locus.

In vitro and in vivo evaluation of 28DAP010, a novel diamidine for treatment of second-stage African sleeping sickness.

African sleeping sickness is a neglected tropical disease transmitted by tsetse flies. New and better drugs are still needed especially for its second stage, which is fatal if untreated. 28DAP010, a dipyridylbenzene analogue of DB829, is the second simple diamidine found to cure mice with central nervous system infections by a parenteral route of administration. 28DAP010 showed efficacy similar to that of DB829 in dose-response studies in mouse models of first- and second-stage African sleeping sickness. The in vitro time to kill, determined by microcalorimetry, and the parasite clearance time in mice were shorter for 28DAP010 than for DB829. No cross-resistance was observed between 28DAP010 and pentamidine on the tested Trypanosoma brucei gambiense isolates from melarsoprol-refractory patients. 28DAP010 is the second promising preclinical candidate among the diamidines for the treatment of second-stage African sleeping sickness.

Pharmacokinetics and pharmacodynamics utilizing unbound target tissue exposure as part of a disposition-based rationale for lead optimization of benzoxaboroles in the treatment of Stage 2 Human African Trypanosomiasis.

SUMMARY This review presents a progression strategy for the discovery of new anti-parasitic drugs that uses in vitro susceptibility, time-kill and reversibility measures to define the therapeutically relevant exposure required in target tissues of animal infection models. The strategy is exemplified by the discovery of SCYX-7158 as a potential oral treatment for stage 2 (CNS) Human African Trypanosomiasis (HAT). A critique of current treatments for stage 2 HAT is included to provide context for the challenges of achieving target tissue disposition and the need for establishing pharmacokinetic-pharmacodynamic (PK-PD) measures early in the discovery paradigm. The strategy comprises 3 stages. Initially, compounds demonstrating promising in vitro activity and selectivity for the target organism over mammalian cells are advanced to in vitro metabolic stability, barrier permeability and tissue binding assays to establish that they will likely achieve and maintain therapeutic concentrations during in-life efficacy studies. Secondly, in vitro time-kill and reversibility kinetics are employed to correlate exposure (based on unbound concentrations) with in vitro activity, and to identify pharmacodynamic measures that would best predict efficacy. Lastly, this information is used to design dosing regimens for pivotal pharmacokinetic-pharmacodyamic studies in animal infection models.

Target-based drug discovery for human African trypanosomiasis: selection of molecular target and chemical matter.

Target-based approaches for human African trypanosomiasis (HAT) and related parasites can be a valuable route for drug discovery for these diseases. However, care needs to be taken in selection of both the actual drug target and the chemical matter that is developed. In this article, potential criteria to aid target selection are described. Then the physiochemical properties of typical oral drugs are discussed and compared to those of known anti-parasitics.

Pharmacokinetics, Trypanosoma brucei gambiense efficacy, and time of drug action of DB829, a preclinical candidate for treatment of second-stage human African trypanosomiasis.

Human African trypanosomiasis (HAT, also called sleeping sickness), a neglected tropical disease endemic to sub-Saharan Africa, is caused by the parasites Trypanosoma brucei gambiense and T. brucei rhodesiense. Current drugs against this disease have significant limitations, including toxicity, increasing resistance, and/or a complicated parenteral treatment regimen. DB829 is a novel aza-diamidine that demonstrated excellent efficacy in mice infected with T. b. rhodesiense or T. b. brucei parasites. The current study examined the pharmacokinetics, in vitro and in vivo activity against T. b. gambiense, and time of drug action of DB829 in comparison to pentamidine. DB829 showed outstanding in vivo efficacy in mice infected with parasites of T. b. gambiense strains, despite having higher in vitro 50% inhibitory concentrations (IC50s) than against T. b. rhodesiense strain STIB900. A single dose of DB829 administered intraperitoneally (5 mg/kg of body weight) cured all mice infected with different T. b. gambiense strains. No cross-resistance was observed between DB829 and pentamidine in T. b. gambiense strains isolated from melarsoprol-refractory patients. Compared to pentamidine, DB829 showed a greater systemic exposure when administered intraperitoneally, partially contributing to its improved efficacy. Isothermal microcalorimetry and in vivo time-to-kill studies revealed that DB829 is a slower-acting trypanocidal compound than pentamidine. A single dose of DB829 (20 mg/kg) administered intraperitoneally clears parasites from mouse blood within 2 to 5 days. In summary, DB829 is a promising preclinical candidate for the treatment of first- and second-stage HAT caused by both Trypanosoma brucei subspecies.

Isolation of Trypanosoma brucei gambiense from cured and relapsed sleeping sickness patients and adaptation to laboratory mice.

BACKGROUND: Sleeping sickness due to Trypanosoma brucei (T.b.) gambiense is still a major public health problem in some central African countries. Historically, relapse rates around 5% have been observed for treatment with melarsoprol, widely used to treat second stage patients. Later, relapse rates of up to 50% have been recorded in some isolated foci in Angola, Sudan, Uganda and Democratic Republic of the Congo (DRC). Previous investigations are not conclusive on whether decreased sensitivity to melarsoprol is responsible for these high relapse rates. Therefore we aimed to establish a parasite collection isolated from cured as well as from relapsed patients for downstream comparative drug sensitivity profiling. A major constraint for this type of investigation is that T.b. gambiense is particularly difficult to isolate and adapt to classical laboratory rodents. METHODOLOGY/PRINCIPAL FINDINGS: From 360 patients treated in Dipumba hospital, Mbuji-Mayi, D.R. Congo, blood and cerebrospinal fluid (CSF) was collected before treatment. From patients relapsing during the 24 months follow-up, the same specimens were collected. Specimens with confirmed parasite presence were frozen in liquid nitrogen in a mixture of Triladyl, egg yolk and phosphate buffered glucose solution. Isolation was achieved by inoculation of the cryopreserved specimens in Grammomys surdaster, Mastomys natalensis and SCID mice. Thus, 85 strains were isolated from blood and CSF of 55 patients. Isolation success was highest in Grammomys surdaster. Forty strains were adapted to mice. From 12 patients, matched strains were isolated before treatment and after relapse. All strains belong to T.b. gambiense type I. CONCLUSIONS AND SIGNIFICANCE: We established a unique collection of T.b. gambiense from cured and relapsed patients, isolated in the same disease focus and within a limited period. This collection is available for genotypic and phenotypic characterisation to investigate the mechanism behind abnormally high treatment failure rates in Mbuji-Mayi, D.R. Congo.

Polysomnography as a diagnosis and post-treatment follow-up tool in human African trypanosomiasis: a case study in an infant.

Gambian (Trypanosoma brucei gambiense) human African trypanosomiasis (HAT) evolves from the hemolymphatic stage 1, treated with pentamidine, to the meningoencephalitic stage 2, often treated with melarsoprol. This arseniate may provoke a deadly reactive encephalopathy. It is therefore crucial to diagnose precisely the stages of HAT, especially when clinical and biological examinations are doubtful. We present here the case of a 30-month old girl (E20 KOLNG) diagnosed with stage 1 HAT during a field survey in June 2007 in Congo. She was followed-up every six months for 18 months in a village dispensary facility at Mpouya. Her health status deteriorated in December 2008, although cerebrospinal fluid (CSF) white blood cell (WBC) count was normal. The child was hospitalized at Brazzaville and a daytime polysomnographic recording (electroencephalogram, electrooculogram, and electromyogram) was performed (Temec Vitaport 3® portable recorder) to avoid a new lumbar puncture. The child presented a complete polysomnographic syndrome of HAT with a major disturbance of the distribution of sleep and wake episodes and the occurrence of sleep onset REM periods (SOREMPs). The relapse at stage 2 was confirmed by a new CSF examination that showed an elevated WBC count (23cells·µL(-1)) with the presence of B lymphocytes. Melarsoprol treatment was undertaken. A post-treatment recording was immediately performed, showing the resolution of sleepwake pattern abnormalities. Another polysomnography, taken four months later, confirmed the normalization of sleep-wake patterns indicating healing. We therefore propose that polysomnography, being a non-invasive technique, should be used in children to alleviate burden caused by HAT staging procedures, especially regarding lumbar punctures in remote African villages.

Trypanosoma brucei gambiense: HMI-9 medium containing methylcellulose and human serum supports the continuous axenic in vitro propagation of the bloodstream form.

Trypanosoma brucei (T.b.) gambiense causes the chronic form of human African trypanosomiasis or sleeping sickness. One of the major problems with studying T.b. gambiense is the difficulty to isolate it from its original host and the difficult adaptation to in vivo and in vitro mass propagation. The objective of this study was to evaluate if an established method for axenic culture of pleomorphic bloodstream form T.b. brucei strains, based on methylcellulose containing HMI-9 medium, also facilitated the continuous in vitro propagation of other bloodstream form Trypanozoon strains, in particular of T.b. gambiense. Bloodstream form trypanosomes from one T.b. brucei, two T.b. rhodesiense, one T. evansi and seven T.b. gambiense strains were isolated from mouse blood and each was concurrently cultivated in liquid and methylcellulose-containing HMI-9 based medium, either with or without additional human serum supplementation, for over 10 consecutive sub passages. Although HMI-9 based medium supplemented with 1.1% (w/v) methylcellulose supported the continuous cultivation of all non-gambiense strains better than liquid media could, the in vitro cultivation of all gambiense strains was only achieved in HMI-9 based medium containing 1.1% (w/v) methylcellulose, 15% (v/v) fetal calf serum and 5% (v/v) heat-inactivated human serum.

Monitoring the pleomorphism of Trypanosoma brucei gambiense isolates in mouse: impact on its transmissibility to Glossina palpalis gambiensis.

Substantial differences have been observed between the cyclical transmission of three Trypanosoma brucei gambiense field isolates in Glossina palpalis gambiensis (Ravel et al., 2006). Differences in the pleomorphism of these isolates in rodent used to provide the infective feed to Glossina, could explain such results, since stumpy forms are preadapted for differentiation to procyclic forms when taken up in a tsetse bloodmeal. To assess this possibility, mice were immunosuppressed and inoculated intraperitoneally with the three isolates (six mice for each trypanosome isolate); then parasitaemia and pleomorphism were determined daily for each mouse. The three T. b. gambiense isolates induced different infection patterns in mouse. The parasitaemia peak was rapidly reached for all the isolates and maintained until mice death for two isolates, while the third isolate rapidly showed a falling phase followed by a second parasitaemia plateau. The proportion of the stumpy forms varied from 15% to 70% over the duration of the experiment and according to the isolate. One isolate, which displayed the highest proportion of stumpy forms and reached the stumpy peak at the onset of the falling phase of parasitaemia, was used to study the relationship between the proportion of stumpy forms and transmissibility to tsetse fly. The results indicated that the transmissibility of trypanosomes was not correlated to the proportion of non-dividing stumpy forms.

Virulence and pathogenicity patterns of Trypanosoma brucei gambiense field isolates in experimentally infected mouse: differences in host immune response modulation by secretome and proteomics.

Human African trypanosomiasis is characterised by an important clinical diversity. Although Trypanosoma brucei gambiense field stocks isolated from patients in the same focus did not exhibit apparent genetic variability, they showed marked differences in terms of virulence (capacity to multiply inside a host) and pathogenicity (ability of producing mortality) in experimental murine infections. Two strains exhibiting opposite pathogenic and virulence properties in mouse were further investigated through their host-parasite interactions. In vitro, parasite bloodstream forms or soluble factors (or secretome) from both strains induced macrophage arginase as a function of their virulence. Arginase expression, a hallmark of macrophage alternative activation pathway, favours trypanosome bloodstream forms development. Moreover, a comparative proteomic study of the trypanosome stocks' secretomes evidenced both a differential expression of common molecules and the existence of stock specific molecules. This highlighted the potential involvement of the differential expression of the same genome in the diverse infectious properties of trypanosomes.

Clinical presentation and treatment outcome of sleeping sickness in Sudanese pre-school children.

BACKGROUND: Existing data on human African trypanosomiasis (HAT) due to Trypanosoma brucei gambiense among children are limited. Here, we described the demographic, clinical, diagnostic, treatment and outcome characteristics of HAT in pre-school children from Kajo-Keji County, South Sudan in comparison with older patients. METHODS: We did a retrospective analysis of HAT patients treated at the Kiri Sleeping Sickness Treatment Centre (SSTC), Kajo-Keji County, from June 2000 to December 2002. RESULTS: Of 1958 HAT patients, 119 (6.1%) were pre-school children (<6 years) including 56 (47%) in first-stage illness and 63 (53%) in second-stage. The proportion of children in second-stage HAT was significantly higher in very young children (<2 years). Walking and speech disturbances were more frequent in second-stage HAT but other neurological symptoms and signs were not associated with disease stage. Pentamidine treatment for first-stage illness was very safe and effective among pre-school children. In contrast, 4.9% of pre-school children in second-stage illness died during melarsoprol treatment and 46% had > or = 1 severe adverse event(s). Macular rash, jaundice and skin necrosis on injection site were significantly more frequent in this age group (p<0.05). Melarsoprol-induced encephalopatic syndrome was less frequent but more severe than in older age groups. CONCLUSION: The clinical features of T. b. gambiense HAT among pre-school children are insufficiently stage-specific. Therefore, laboratory-based staging is mandatory to prevent unnecessary harm to HAT patients caused by the high toxicity of melarsoprol.

Diamidines as antitrypanosomal, antileishmanial and antimalarial agents.

Diamidine-containing compounds have a long history of use in the treatment of African trypanosomiasis and leishmaniasis. The discovery that diamidine prodrugs possess in vivo antimicrobial activity when administered orally has led to a renewed interest in this class of compounds for the treatment of parasitic infections. In this review, the selectivity of diamidines against trypanosomes, Leishmania and Plasmodium is rationalized through mechanism-of-action studies. An overview of the antiprotozoal activities of newer diamidines and diamidine prodrugs is also presented, along with a summary of the progress made toward the clinical development of new diamidines for use against these parasitic diseases.

Prospective analysis for community participation in trypanosomosis control in The Gambia.

The shift towards community participation in the eradication of trypanosomosis calls for investigation the underlying incentive structure for individuals in the community to cooperate in the provision of various control methods. Survey data were used to assess patterns of the community's demand for insecticide pour-ons and trypanocidal drugs and factors affecting individual demand in The Gambia. The results show that insecticide pour-on is strongly preferred. Similarly, farmers revealed a preference for community-based provision scheme. Factors affecting an individual farmer's decision to invest in either pour-on or trypanocidal drugs were highlighted. While there are many factors associated with farmer's decisions to invest in trypanosomosis control methods and to participate in collective actions, the results indicate that farmers are ready to anticipate complete privatization of veterinary services through community-based schemes.

Spontaneous cure of domestic pigs experimentally infected by Trypanosoma brucei gambiense. Implications for the control of sleeping sickness.

The existence of a pig reservoir for human African trypanosomosis (HAT) due to Trypanosoma brucei gambiense complicates the fight against this disease. This study, reports results obtained from pigs, which were inoculated with the blood of a person, suffering from HAT in Cameroon. The pigs were reared and kept in the shelter from all contact with Glossina, and monitored for 188 days. The seroconversion was checked by agglutination assays for trypanosomosis (CATT 1.3 and LATEX/T.b.gambiense). The parasitemia was measured by quantitative buffy coat method (QBC) and by polymerase chain reaction method (PCR). In addition, growth was recorded as well as blood counting and blood formulas. The results showed that the pigs were trypanotolerant and cure themselves in less than 6 months. It is concluded that sterilisation of this reservoir could be achieved by tsetse-control measures in 1 year. It confirms the strategy to complement screening and treatment of HAT with tsetse fly control measures.

Effects of heparin administration on Trypanosoma brucei gambiense infection in rats.

We examined whether heparin administration influences in vivo trypanosome proliferation in infected rats. Administration of heparin every 8 hr via cardiac catheter inhibited growth of Trypanosoma brucei gambiense and prolonged survival of treated rats. Heparin administration increased lipoprotein lipase activity, high-density lipoprotein (HDL) concentration in the blood, and haptoglobin messenger RNA content of the liver. The presence of heparin in culture media did not directly affect proliferation of trypanosomes in vitro. However, the addition of plasma from infected rats treated with heparin to culture media decreased the number of trypanosomes. This effect was decreased by incubating the trypanosomes with benzyl alcohol, a known inhibitor of receptor-mediated endocytosis of lipoprotein. These data suggested that heparin administration reduced the number of trypanosomes in infected rats. Trypanosome lytic factor, a HDL and haptoglobin-related protein, protects humans and some animals from infection by Trypanosoma brucei brucei. In rats, increases in HDL and haptoglobin may affect the proliferation of T. b. gambiense.

Sleep structure: a new diagnostic tool for stage determination in sleeping sickness.

Human African trypanosomiasis (HAT), due to the transmission of Trypanosoma brucei (T. b.) gambiense and T. b. rhodesiense by tsetse flies, is re-emerging in inter-tropical Africa. It evolves from the hemolymphatic Stage I to the meningo-encephalitic Stage II. The latter is generally treated with melarsoprol, an arseniate provoking often a deadly encephalopathy. A precise determination of the HAT evolution stage is therefore crucial. Stage II patients show: (i) a deregulation of the 24-h distribution of the sleep-wake alternation; (ii) an alteration of the sleep structure, with frequent sleep onset rapid eye movement (REM) periods (SOREMPs). Gambian HAT was diagnosed in eight patients (four, Stage II; three, Stage I; one, "intermediate" case) at the trypanosomiasis clinic at Viana (Angola). Continuous 48-h polysomnography was recorded on Oxford Medilog 9000-II portable systems before and after treatment with melarsoprol (Stage II) or pentamidine (Stage I and "intermediate" stage). Sleep traces were visually analyzed in 20-s epochs using the PRANA software. Stage II patients showed the complete sleep-wake syndrome, partly reversed by melarsoprol 1 month later. Two Stage I patients did not experience any of these alterations. However, the "intermediate" and one Stage I patients exhibited sleep disruptions and/or SOREMPs, persistent after pentamidine treatment. Polysomnography may represent a diagnostic tool to distinguish the two stages of HAT. Especially, SOREMPs appear shortly after the central nervous system invasion by trypanosomes. The reversibility of the sleep-wake cycle and sleep structure alterations after appropriate treatment constitutes the basis of an evaluation of the healing process.

History of sleeping sickness (African trypanosomiasis).

Infections with subspecies of the protozoan parasite Trypanosoma brucei cause important wasting diseases in Africa (nagana in cattle and sleeping sickness in humans). These diseases were little known until the end of the nineteenth century when serious epidemics of nagana were reported and raised concern among the colonial powers. The early history of sleeping sickness revolves around the discovery of the causative organism, its mode of transmission,and its life cycle in the tsetse fly. The history continues into the twentieth century with the discovery of how the parasites evade the immune response, frustrating the development of a vaccine; the failure to develop cheap and effective drugs; and the development of alternative approaches to control the tsetse fly vector.

Spatial and temporal patterns of human African trypanosomosis (HAT) transmission risk in the Bipindi focus, in the forest zone of southern Cameroon.

Vector control is an effective and cost-efficient way to disrupt the transmission of human African trypanosomosis (HAT); it has nonetheless been little used to date in the disease's foci. With the aim to target trapping more precisely and to develop an optimized vector control system, a transmission risk index was used in the HAT focus of Bipindi, in the forest zone of southern Cameroon. The authors used a simplified version of the index originally developed by Laveissière et al. in 1994. The calculation of this new index only requires knowledge of the proportion of teneral flies and the proportion of flies with human blood meals in samples caught in different biotopes. This makes it possible to identify the biotopes displaying permanent risk, such as riverbanks, as well as biotopes displaying seasonal risk, such as marshy hollows and encampmemts. In the villages, the domestic pig, with 49% of the identified blood meals, is the favorite host of the tsetse flies during the short rainy season. The proportion of blood meals taken on human beings does not significantly increase when domestic pigs are absent. Game animals, contributing to 46% and 64% of the blood meals during the short rainy season and the long dry season, respectively, are also favored as feeding hosts in this particular HAT focus.