Model Of Human African Trypanosomiasis Research Articles

Chemical Optimization of CBL0137 for Human African Trypanosomiasis Lead Drug Discovery.

The carbazole CBL0137 (1) is a lead for drug development against human African trypanosomiasis (HAT), a disease caused by Trypanosoma brucei. To advance 1 as a candidate drug, we synthesized new analogs that were evaluated for the physicochemical properties, antitrypanosome potency, selectivity against human cells, metabolism in microsomes or hepatocytes, and efflux ratios. Structure-activity/property analyses of analogs revealed eight new compounds with higher or equivalent selectivity indices (5j, 5t, 5v, 5w, 5y, 8d, 13i, and 22e). Based on the overall compound profiles, compounds 5v and 5w were selected for assessment in a mouse model of HAT; while 5v demonstrated a lead-like profile for HAT drug development, 5w showed a lack of efficacy. Lessons from these studies will inform further optimization of carbazoles for HAT and other indications.

Fast acting allosteric phosphofructokinase inhibitors block trypanosome glycolysis and cure acute African trypanosomiasis in mice

The parasitic protist Trypanosoma brucei is the causative agent of Human African Trypanosomiasis, also known as sleeping sickness. The parasite enters the blood via the bite of the tsetse fly where it is wholly reliant on glycolysis for the production of ATP. Glycolytic enzymes have been regarded as challenging drug targets because of their highly conserved active sites and phosphorylated substrates. We describe the development of novel small molecule allosteric inhibitors of trypanosome phosphofructokinase (PFK) that block the glycolytic pathway resulting in very fast parasite kill times with no inhibition of human PFKs. The compounds cross the blood brain barrier and single day oral dosing cures parasitaemia in a stage 1 animal model of human African trypanosomiasis. This study demonstrates that it is possible to target glycolysis and additionally shows how differences in allosteric mechanisms may allow the development of species-specific inhibitors to tackle a range of proliferative or infectious diseases.

Hit-to-Lead Optimization of Benzoxazepinoindazoles As Human African Trypanosomiasis Therapeutics.

Human African trypanosomiasis (HAT) is a neglected tropical disease caused by infection with either of two subspecies of the parasite Trypanosoma brucei. Due to a lack of economic incentive to develop new drugs, current treatments have severe limitations in terms of safety, efficacy, and ease of administration. In an effort to develop new HAT therapeutics, we report the structure-activity relationships around T. brucei for a series of benzoxazepinoindazoles previously identified through a high-throughput screen of human kinase inhibitors, and the subsequent in vivo experiments for HAT. We identified compound 18, which showed an improved kinase selectivity profile and acceptable pharmacokinetic parameters, as a promising lead. Although treatment with 18 cured 60% of mice in a systemic model of HAT, the compound was unable to clear parasitemia in a CNS model of the disease. We also report the results of cross-screening these compounds against T. cruzi, L. donovani, and S. mansoni.

Generation of neuroinflammation in human African trypanosomiasis.

Human African trypanosomiasis (HAT) is caused by infection due to protozoan parasites of the Trypanosoma genus and is a major fatal disease throughout sub-Saharan Africa. After an early hemolymphatic stage in which the peripheral tissues are infected, the parasites enter the CNS causing a constellation of neurologic features. Although the CNS stage of HAT has been recognized for over a century, the mechanisms generating the neuroinflammatory response are complex and not well understood. Therefore a better understanding of the mechanisms utilized by the parasites to gain access to the CNS compartment is critical to explaining the generation of neuroinflammation. Contrast-enhanced MRI in a murine model of HAT has shown an early and progressive deterioration of blood-CNS barrier function after trypanosome infection that can be reversed following curative treatment. However, further studies are required to clarify the molecules involved in this process. Another important determinant of brain inflammation is the delicate balance of proinflammatory and counterinflammatory mediators. In mouse models of HAT, proinflammatory mediators such as tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and CXCL10 have been shown to be crucial to parasite CNS invasion while administration of interleukin (IL)-10, a counter inflammatory molecule, reduces the CNS parasite burden as well as the severity of the neuroinflammatory response and the clinical symptoms associated with the infection. This review focuses on information, gained from both infected human samples and animal models of HAT, with an emphasis on parasite CNS invasion and the development of neuroinflammation.

Cerebral inducible nitric oxide synthase protein expression in microglia, astrocytes and neurons in Trypanosoma brucei brucei-infected rats.

To study the anatomo-biochemical substrates of brain inflammatory processes, Wistar male rats were infected with Trypanosoma brucei brucei. With this reproducible animal model of human African trypanosomiasis, brain cells (astrocytes, microglial cells, neurons) expressing the inducible nitric oxide synthase (iNOS) enzyme were revealed. Immunohistochemistry was achieved for each control and infected animal through eight coronal brain sections taken along the caudorostral axis of the brain (brainstem, cerebellum, diencephalon and telencephalon). Specific markers of astrocytes (anti-glial fibrillary acidic protein), microglial cells (anti-integrin alpha M) or neurons (anti-Neuronal Nuclei) were employed. The iNOS staining was present in neurons, astrocytes and microglial cells, but not in oligodendrocytes. Stained astrocytes and microglial cells resided mainly near the third cavity in the rostral part of brainstem (periaqueductal gray), diencephalon (thalamus and hypothalamus) and basal telencephalon. Stained neurons were scarce in basal telencephalon, contrasting with numerous iNOS-positive neuroglial cells. Contrarily, in dorsal telencephalon (neocortex and hippocampus), iNOS-positive neurons were plentiful, contrasting with the marked paucity of labelled neuroglial (astrocytes and microglial) cells. The dual distribution between iNOS-labelled neuroglial cells and iNOS-labelled neurons is a feature that has never been described before. Functionalities attached to such a divergent distribution are discussed.

Series of Alkynyl-Substituted Thienopyrimidines as Inhibitors of Protozoan Parasite Proliferation.

Discovery of new chemotherapeutic lead agents can be accelerated by optimizing chemotypes proven to be effective in other diseases to act against parasites. One such medicinal chemistry campaign has focused on optimizing the anilinoquinazoline drug lapatinib (1) and the alkynyl thieno[3,2-d]pyrimidine hit GW837016X (NEU-391, 3) into leads for antitrypanosome drugs. We now report the structure-activity relationship studies of 3 and its analogs against Trypanosoma brucei, which causes human African trypanosomiasis (HAT). The series was also tested against Trypanosoma cruzi, Leishmania major, and Plasmodium falciparum. In each case, potent antiparasitic hits with acceptable toxicity margins over mammalian HepG2 and NIH3T3 cell lines were identified. In a mouse model of HAT, 3 extended life of treated mice by 50%, compared to untreated controls. At the cellular level, 3 inhibited mitosis and cytokinesis in T. brucei. Thus, the alkynylthieno[3,2-d]pyrimidine chemotype is an advanced hit worthy of further optimization as a potential chemotherapeutic agent for HAT.

Cerebrospinal fluid neopterin and CXCL13 are suitable biomarkers for staging and detection of treatment failure in a non-human primate model of human African trypanosomiasis

Abstract Background Follow-up of treated human African trypanosomiasis (HAT) patients has been a routine practice for confirming cure and early detection of treatment failure. Objective The aim of this study was to verify suitability of neopterin and CXCL13 as disease stage biomarkers and monitors of treatment efficacy in a vervet monkey model of HAT. Methods Six monkeys were infected with Trypanosoma brucei. Late stage disease was induced by sub-curative treatment with diminazene aceturate (DA) from 28 days post-infection (dpi). Upon relapse, the animals were treated curatively with melarsoprol (Mel B) from 82 dpi. Blood and cerebrospinal fluid (CSF) samples were collected weekly and assessed for parasitosis, as well as tested for neopterin and CXCL13 by ELISA, for a period of 39 weeks. Results Neopterin concentrations in serum increased rapidly after infection in early disease (stage 1), peaking at 14 dpi. Levels then dropped rapidly to pre-infection levels by 35 dpi. In contrast, there was a marginal increase in CSF neopterin during early infection, with a minor peak at 21 dpi. Serum CXCL13 increased rapidly from 7 dpi, peaking at 28 dpi. Levels dropped upon sub-curative treatment with DA. CXCL13 concentrations in CSF also increased gradually in early stage disease and continued to rise after sub-curative treatment with DA. Curative treatment with Mel B resulted in its gradual decline, reaching pre-infection levels 105 days later. Conclusions CSF neopterin and CXCL13 peak levels coinciding with time of relapses demonstrates the potential of these biomarkers in staging and detecting treatment failure. Moreover, the rapid fall in CSF neopterin after curative treatment confirms its great potential for use in development of a test of cure.

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.

Synthesis and evaluation of analogs of 5′-(((Z)-4-amino-2-butenyl)methylamino)-5′-deoxyadenosine (MDL 73811, or AbeAdo) – An inhibitor of S-adenosylmethionine decarboxylase with antitrypanosomal activity

We describe our efforts to improve the pharmacokinetic properties of a mechanism-based suicide inhibitor of the polyamine biosynthetic enzyme S-adenosylmethionine decarboxylase (AdoMetDC), essential for the survival of the eukaryotic parasite Trypanosoma brucei responsible for Human African Trypanosomiasis (HAT). The lead compound, 5′-(((Z)-4-amino-2-butenyl)methylamino)-5′-deoxyadenosine (1, also known as MDL 73811, or AbeAdo), has curative efficacy at a low dosage in a hemolymphatic model of HAT but displayed no demonstrable effect in a mouse model of the CNS stage of HAT due to poor blood–brain barrier permeation. Therefore, we prepared and evaluated an extensive set of analogs with modifications in the aminobutenyl side chain, the 5′-amine, the ribose, and the purine fragments. Although we gained valuable structure–activity insights from this comprehensive dataset, we did not gain traction on improving the prospects for CNS penetration while retaining the potent antiparasitic activity and metabolic stability of the lead compound 1.

1-Benzyl-3-aryl-2-thiohydantoin Derivatives as New Anti-Trypanosoma brucei Agents: SAR and in Vivo Efficacy.

A high throughput screening and subsequent hit validation identified compound 1 as an inhibitor of Trypanosoma brucei parasite growth. Extensive structure–activity relationship optimization based on antiparasitic activity led to the highly potent compounds, 1-(4-fluorobenzyl)-3-(4-dimethylamino-3-chlorophenyl)-2-thiohydantoin (68) and 1-(2-chloro-4-fluorobenzyl)-3-(4-dimethylamino-3-methoxyphenyl)-2-thiohydantoin (76), with a T. brucei EC50 of 3 and 2 nM, respectively. This represents >100-fold improvement in potency compared to compound 1. In vivo efficacy experiments of 68 and 76 in an acute mouse model of Human African Trypanosomiasis showed a 100% cure rate after 4 days of oral treatment at 50 mg/kg twice per day.

Delineating neuroinflammation, parasite CNS invasion, and blood-brain barrier dysfunction in an experimental murine model of human African trypanosomiasis.

Although Trypanosoma brucei spp. was first detected by Aldo Castellani in CSF samples taken from sleeping sickness patients over a century ago there is still a great deal of debate surrounding the timing, route and effects of transmigration of the parasite from the blood to the CNS. In this investigation, we have applied contrast-enhance magnetic resonance imaging (MRI) to study the effects of trypanosome infection on the blood-brain barrier (BBB) in the well-established GVR35 mouse model of sleeping sickness. In addition, we have measured the trypanosome load present in the brain using quantitative Taqman PCR and assessed the severity of the neuroinflammatory reaction at specific time points over the course of the infection. Contrast enhanced-MRI detected a significant degree of BBB impairment in mice at 14days following trypanosome infection, which increased in a step-wise fashion as the disease progressed. Parasite DNA was present in the brain tissue on day 7 after infection. This increased significantly in quantity by day 14 post-infection and continued to rise as the infection advanced. A progressive increase in neuroinflammation was detected following trypanosome infection, reaching a significant level of severity on day 14 post-infection and rising further at later time-points. In this model stage-2 disease presents at 21days post-infection. The combination of the three methodologies indicates that changes in the CNS become apparent prior to the onset of established stage-2 disease. This could in part account for the difficulties associated with defining specific criteria to distinguish stage-1 and stage-2 infections and highlights the need for improved staging diagnostics.

Dose-dependent effect and pharmacokinetics of fexinidazole and its metabolites in a mouse model of human African trypanosomiasis

Human African trypanosomiasis (HAT) is a neglected tropical disease, with a population of 70 million at risk. Current treatment options are limited. In the search for new therapeutics, the repurposing of the broad-spectrum antiprotozoal drug fexinidazole has completed Phase III trials with the anticipation that it will be the first oral treatment for HAT.This study used the recently validated bioluminescence imaging model to assess the dose and rate of kill effect of fexinidazole in infected mice, and the dose-dependent effect of fexinidazole on trypanosome infection. Pharmacokinetics of fexinidazole in plasma and central nervous system (CNS) compartments were similar in both infected and uninfected mice. Drug distribution within the CNS was further examined by microdialysis, showing similar levels in the cortex and hippocampus. However, high variability in drug distribution and exposure was found between mice.

IgM, lgG and IL-6 profiles in the Trypanosoma brucei brucei monkey model of human African trypanosomiasis

Human African trypanosomiasis (HAT) patients manifest immunological profiles, whose variations over time can be used to indicate disease progression. However, monitoring of these biomarkers in human patients is beset by several limitations which can be offset by using chronic animal models. A recent improved monkey model of HAT using a Trypanosoma brucei brucei isolate has been developed but the immunological profile has not been elucidated. The objectives of the current study was to determine the IgM, IgG and IL-6 profiles in blood and cerebrospinal fluid (CSF) in vervet monkeys infected with T. b. brucei. Three vervet monkeys were infected intravenously with 105T. b. brucei, monitored for disease development and subsequently treated 28days post infection (dpi) sub-curatively using diminazene aceturate (DA) to induce late stage disease and curatively treated with melarsoprol (Mel B) at 119 dpi, respectively. Matched serum and cerebrospinal fluid (CSF) samples were obtained at regular intervals and immunospecific IgM, immunoglobulin G (IgG) were quantified by ELISA while IL-6 was assayed using a cytometric bead array (CBA) kit.Results showed that following infection, CSF IgM, IgG, IL-6 and serum IL-6 were significantly (p<0.05) elevated with peak levels coinciding with relapse parasitaemia. The IgG levels increased to reach OD peak levels of 0.442±0.5 at 126 dpi. After curative treatment with MelB, the serum IgM and Ig G levels fell rapidly to attain pre-infection levels within 35 and 49days, respectively. This shows that the profile of these immunoglobulins can be used as an indicator of curative treatment. CSF IL-6 concentrations of infected vervet monkeys showed no significant change (P>0.05) between infection and 35 dpi but levels increased significantly (P<0.05) with the highest level of 55.53pg/ml recorded at112 dpi. IL-6 elevation from 35 dpi may be indicative of parasite neuroinvasion hence can be used as possible candidate marker for late stage disease in the monkey model. Further, the marker can also be used in conjunction with IgG and IgM as markers for development of test of cure for HAT.

Cerebrospinal Fluid Neopterin and CXCL13 are Suitable Biomarkers for Staging and Detection of Treatment Failure in a Non-Human Primate Model of Human African Trypanosomiasis

Background: Follow-up of treated human African trypanosomiasis (HAT) patients has been a routine practice for confirming cure and early detection of treatment failure. Objective: The aim of this study was to verify whether the neopterin and CXCL13 biomarkers are suitable for determining the stage of disease, and for monitoring the efficacy of treatment in a vervet monkey model of HAT. Methods: Six monkeys were infected with Trypanosoma brucei. Late stage disease was induced by sub-curative treatment with diminazene aceturate (DA) from 28 days’ post-infection (dpi). When relapses occurred, the animals were treated curatively with melarsoprol (Mel B) from 82 dpi. Blood and cerebrospinal fluid (CSF) samples were collected at weekly intervals and assessed for parasitosis, as well as tested for neopterin and CXCL13 by ELISA, for a period of 39 weeks. Results: The concentration of neopterin in serum increased rapidly after infection in early disease (stage 1), peaking at 14 dpi. Levels then dropped rapidly to pre-infection levels by 35 dpi. In contrast, there was a marginal increase in CSF neopterin during early infection, with a minor peak at 21 dpi. Serum CXCL13 increased rapidly from 7 dpi, peaking at 28 dpi, after which the levels dropped upon sub-curative treatment with DA. The concentration of CXCL13 in CSF also increased gradually in early stage disease and continued to rise after sub-curative treatment with DA. Curative treatment with Mel B resulted in its gradual decline, reaching pre-infection levels 105 days later. Conclusions: The changes in CSF neopterin and CXCL13 correspond to important time-points and stages of the disease. That peak levels coincided with time of relapses demonstrates the potential of these biomarkers in staging and detecting treatment failure. Moreover, the rapid fall in CSF neopterin after curative treatment confirms its great potential for use in development of a test of cure.

Discovery of a Carbazole-Derived Lead Drug for Human African Trypanosomiasis.

The protozoan parasite Trypanosoma brucei causes the fatal illness human African trypanosomiasis (HAT). Standard of care medications currently used to treat HAT have severe limitations, and there is a need to find new chemical entities that are active against infections of T. brucei. Following a “drug repurposing” approach, we tested anti-trypanosomal effects of carbazole-derived compounds called “Curaxins”. In vitro screening of 26 compounds revealed 22 with nanomolar potency against axenically cultured bloodstream trypanosomes. In a murine model of HAT, oral administration of compound 1 cured the disease. These studies established 1 as a lead for development of drugs against HAT. Pharmacological time-course studies revealed the primary effect of 1 to be concurrent inhibition of mitosis coupled with aberrant licensing of S-phase entry. Consequently, polyploid trypanosomes containing 8C equivalent of DNA per nucleus and three or four kinetoplasts were produced. These effects of 1 on the trypanosome are reminiscent of “mitotic slippage” or endoreplication observed in some other eukaryotes.

Discovery of Indoline-2-carboxamide Derivatives as a New Class of Brain-Penetrant Inhibitors of Trypanosoma brucei.

There is an urgent need for new, brain penetrant small molecules that target the central nervous system second stage of human African trypanosomiasis (HAT). We report that a series of novel indoline-2-carboxamides have been identified as inhibitors of Trypanosoma brucei from screening of a focused protease library against Trypanosoma brucei brucei in culture. We describe the optimization and characterization of this series. Potent antiproliferative activity was observed. The series demonstrated excellent pharmacokinetic properties, full cures in a stage 1 mouse model of HAT, and a partial cure in a stage 2 mouse model of HAT. Lack of tolerability prevented delivery of a fully curative regimen in the stage 2 mouse model and thus further progress of this series.

Virulence and pathogenicity of three Trypanosoma brucei rhodesiense stabilates in a Swiss white mouse model.

A key objective in basic research on human African trypanosomiasis (HAT) is developing a cheap and reliable experimental model of the disease for use in pathogenesis and drug studies. With a view to improving current models, a study was undertaken to characterise the virulence and pathogenicity of three Trypanosoma brucei rhodesiense stabilates, labelled as International Livestock Research Institute (ILRI)-2918, ILRI-3953, and Institute of Primate Research (IPR)-001, infected into Swiss white mice. Swiss white mice were infected intraperitoneally with trypanosomes and observed for parasitaemia using wet blood smears obtained by tail snipping. Induction of late-stage disease was undertaken using diminazene aceturate (40 mg/kg, Berenil) with curative treatment done using melarsoprol (3.6 mg/kg, Arsobal). The prepatent period for the stabilates ranged from three to four days with mean peak parasitaemia ranging from Log10 6.40 to 8.36. First peak parasitaemia for all stabilates varied between six and seven days post infection (DPI) followed by secondary latency in ILRI-2918 (15-17 DPI) and IPR-001 (17-19 DPI). Survival times ranged from six DPI (ILRI-3953) to 86 DPI (IPR-001). Hindleg paresis was observed in both ILRI-3953 (at peak parasitaemia) and ILRI-2918 (after relapse parasitaemia). Mice infected with IPR-001 survived until 54 DPI when curative treatment was undertaken. This study demonstrated that the stabilates ILRI-2918 and ILRI-3953 were unsuitable for modelling late-stage HAT in mice. The stabilate IPR-001 demonstrated the potential to induce chronic trypanosomiasis in Swiss white mice for use in development of a late-stage model of HAT.

Lead optimization of a pyrazole sulfonamide series of Trypanosoma brucei N-myristoyltransferase inhibitors: identification and evaluation of CNS penetrant compounds as potential treatments for stage 2 human African trypanosomiasis.

Trypanosoma bruceiN-myristoyltransferase (TbNMT) is an attractive therapeutic target for the treatment of human African trypanosomiasis (HAT). From previous studies, we identified pyrazole sulfonamide, DDD85646 (1), a potent inhibitor of TbNMT. Although this compound represents an excellent lead, poor central nervous system (CNS) exposure restricts its use to the hemolymphatic form (stage 1) of the disease. With a clear clinical need for new drug treatments for HAT that address both the hemolymphatic and CNS stages of the disease, a chemistry campaign was initiated to address the shortfalls of this series. This paper describes modifications to the pyrazole sulfonamides which markedly improved blood–brain barrier permeability, achieved by reducing polar surface area and capping the sulfonamide. Moreover, replacing the core aromatic with a flexible linker significantly improved selectivity. This led to the discovery of DDD100097 (40) which demonstrated partial efficacy in a stage 2 (CNS) mouse model of HAT.

Safety, Pharmacokinetic, and Efficacy Studies of Oral DB868 in a First Stage Vervet Monkey Model of Human African Trypanosomiasis

There are no oral drugs for human African trypanosomiasis (HAT, sleeping sickness). A successful oral drug would have the potential to reduce or eliminate the need for patient hospitalization, thus reducing healthcare costs of HAT. The development of oral medications is a key objective of the Consortium for Parasitic Drug Development (CPDD). In this study, we investigated the safety, pharmacokinetics, and efficacy of a new orally administered CPDD diamidine prodrug, 2,5-bis[5-(N-methoxyamidino)-2-pyridyl]furan (DB868; CPD-007-10), in the vervet monkey model of first stage HAT. DB868 was well tolerated at a dose up to 30 mg/kg/day for 10 days, a cumulative dose of 300 mg/kg. Mean plasma levels of biomarkers indicative of liver injury (alanine aminotransferase, aspartate aminotransferase) were not significantly altered by drug administration. In addition, no kidney-mediated alterations in creatinine and urea concentrations were detected. Pharmacokinetic analysis of plasma confirmed that DB868 was orally available and was converted to the active compound DB829 in both uninfected and infected monkeys. Treatment of infected monkeys with DB868 began 7 days post-infection. In the infected monkeys, DB829 attained a median Cmax (dosing regimen) that was 12-fold (3 mg/kg/day for 7 days), 15-fold (10 mg/kg/day for 7 days), and 31-fold (20 mg/kg/day for 5 days) greater than the IC50 (14 nmol/L) against T. b. rhodesiense STIB900. DB868 cured all infected monkeys, even at the lowest dose tested. In conclusion, oral DB868 cured monkeys with first stage HAT at a cumulative dose 14-fold lower than the maximum tolerated dose and should be considered a lead preclinical candidate in efforts to develop a safe, short course (5–7 days), oral regimen for first stage HAT.

Safety, Pharmacokinetic, and Efficacy Studies of Oral DB868 in a First Stage Vervet Monkey Model of Human African Trypanosomiasis

Safety, Pharmacokinetic, and Efficacy Studies of Oral DB868 in a First Stage Vervet Monkey Model of Human African Trypanosomiasis