Repurposing Pyramax®, quinacrine and tilorone as treatments for Ebola virus disease.

We have recently identified three molecules (tilorone, quinacrine and pyronaridine tetraphosphate) which all demonstrated efficacy in the mouse model of infection with mouse-adapted Ebola virus (EBOV) model of disease and had similar in vitro inhibition of an Ebola pseudovirus (VSV-EBOV-GP), suggesting they interfere with viral entry. Using a machine learning model to predict lysosomotropism these compounds were evaluated for their ability to possess a lysosomotropic mechanism in vitro. We now demonstrate in vitro that pyronaridine tetraphosphate is an inhibitor of Lysotracker accumulation in lysosomes (IC50 = 0.56 µM). Further, we evaluated antiviral synergy between pyronaridine and artesunate (Pyramax®), which are used in combination to treat malaria. Artesunate was not found to have lysosomotropic activity in vitro and the combination effect on EBOV inhibition was shown to be additive. Pyramax® may represent a unique example of the repurposing of a combination product for another disease.

Identification of a 4-fluorobenzyl l-valinate amide benzoxaborole (AN11736) as a potential development candidate for the treatment of Animal African Trypanosomiasis (AAT).

Novel l-valinate amide benzoxaboroles and analogues were designed and synthesized for a structure-activity-relationship (SAR) investigation to optimize the growth inhibitory activity against Trypanosoma congolense (T. congolense) and Trypanosoma vivax (T. vivax) parasites. The study identified 4-fluorobenzyl (1-hydroxy-7-methyl-1,3-dihydrobenzo[c][1,2]oxaborole-6-carbonyl)-l-valinate (5, AN11736), which showed IC50 values of 0.15 nM against T. congolense and 1.3 nM against T. vivax, and demonstrated 100% efficacy with a single dose of 10 mg/kg against both T. congolense and T. vivax in mouse models of infection (IP dosing) and in the target animal, cattle, dosed intramuscularly. AN11736 has been advanced to early development studies.

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.

Application of a resazurin-based high-throughput screening assay for the identification and progression of new treatments for human African trypanosomiasis.

Human African trypanosomiasis (HAT) is caused by the protozoan parasite Trypanosoma brucei, and the disease is fatal if untreated. There is an urgent need to develop new, safe and effective treatments for HAT because current drugs have extremely poor safety profiles and are difficult to administer. Here we report the development and application of a cell-based resazurin reduction assay for high throughput screening and identification of new inhibitors of T. b. brucei as starting points for the development of new treatments for human HAT. Active compounds identified in primary screening of ∼48,000 compounds representing ∼25 chemical classes were titrated to obtain IC50 values. Cytotoxicity against a mammalian cell line was determined to provide indications of parasite versus host cell selectivity. Examples from hit series that showed selectivity and evidence of preliminary SAR were re-synthesized to confirm trypanocidal activity prior to initiating hit-to-lead expansion efforts. Additional assays such as serum shift, time to kill and reversibility of compound effect were developed and applied to provide further criteria for advancing compounds through the hit-to-lead phase of the project. From this initial effort, six distinct chemical series were selected and hit-to-lead chemistry was initiated to synthesize several key analogs for evaluation of trypanocidal activity in the resazurin-reduction assay for parasite viability. From the hit-to-lead efforts, a series was identified that demonstrated efficacy in a mouse model for T. b. brucei infection and was progressed into the lead optimization stage. In summary, the present study demonstrates the successful and effective use of resazurin-reduction based assays as tools for primary and secondary screening of a new compound series to identify leads for the treatment of HAT.

SCYX-7158, an orally-active benzoxaborole for the treatment of stage 2 human African trypanosomiasis.

BACKGROUND: Human African trypanosomiasis (HAT) is an important public health problem in sub-Saharan Africa, affecting hundreds of thousands of individuals. An urgent need exists for the discovery and development of new, safe, and effective drugs to treat HAT, as existing therapies suffer from poor safety profiles, difficult treatment regimens, limited effectiveness, and a high cost of goods. We have discovered and optimized a novel class of small-molecule boron-containing compounds, benzoxaboroles, to identify SCYX-7158 as an effective, safe and orally active treatment for HAT. METHODOLOGY/PRINCIPAL FINDINGS: A drug discovery project employing integrated biological screening, medicinal chemistry and pharmacokinetic characterization identified SCYX-7158 as an optimized analog, as it is active in vitro against relevant strains of Trypanosoma brucei, including T. b. rhodesiense and T. b. gambiense, is efficacious in both stage 1 and stage 2 murine HAT models and has physicochemical and in vitro absorption, distribution, metabolism, elimination and toxicology (ADMET) properties consistent with the compound being orally available, metabolically stable and CNS permeable. In a murine stage 2 study, SCYX-7158 is effective orally at doses as low as 12.5 mg/kg (QD×7 days). In vivo pharmacokinetic characterization of SCYX-7158 demonstrates that the compound is highly bioavailable in rodents and non-human primates, has low intravenous plasma clearance and has a 24-h elimination half-life and a volume of distribution that indicate good tissue distribution. Most importantly, in rodents brain exposure of SCYX-7158 is high, with C(max) >10 µg/mL and AUC(0-24 hr) >100 µg*h/mL following a 25 mg/kg oral dose. Furthermore, SCYX-7158 readily distributes into cerebrospinal fluid to achieve therapeutically relevant concentrations in this compartment. CONCLUSIONS/SIGNIFICANCE: The biological and pharmacokinetic properties of SCYX-7158 suggest that this compound will be efficacious and safe to treat stage 2 HAT. SCYX-7158 has been selected to enter preclinical studies, with expected progression to phase 1 clinical trials in 2011.

SAR of 2-amino and 2,4-diamino pyrimidines with in vivo efficacy against Trypanosoma brucei.

A series of 2,4-diaminopyrimidines was investigated and compounds were found to have in vivo efficacy against Trypanosoma brucei in an acute mouse model. However, in vitro permeability data suggested the 2,4-diaminopyrimidenes would have poor permeability through the blood brain barrier. Consequently a series of 4-desamino analogs were synthesized and found to have improved in vitro permeability.

2,4-Diaminopyrimidines as potent inhibitors of Trypanosoma brucei and identification of molecular targets by a chemical proteomics approach.

BACKGROUND: There is an urgent need to develop new, safe and effective treatments for human African trypanosomiasis (HAT) because current drugs have extremely poor safety profiles and are difficult to administer. Here we report the discovery of 2,4-diaminopyrimidines, exemplified by 4-[4-amino-5-(2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidine-1-carboxylic acid phenylamide (SCYX-5070), as potent inhibitors of Trypanosoma brucei and the related trypanosomatid protozoans Leishmania spp. METHODOLOGY/PRINCIPAL FINDINGS: In this work we show that loss of T. brucei viability following SCYX-5070 exposure was dependent on compound concentration and incubation time. Pulse incubation of T. brucei with SCYX-5070 demonstrates that a short period of exposure (10-12 hrs) is required to produce irreversible effects on survival or commit the parasites to death. SCYX-5070 cured an acute trypanosomiasis infection in mice without exhibiting signs of compound related acute or chronic toxicity. To identify the molecular target(s) responsible for the mechanism of action of 2,4-diaminopyrimidines against trypanosomatid protozoa, a representative analogue was immobilized on a solid matrix (sepharose) and used to isolate target proteins from parasite extracts. Mitogen-activated protein kinases (MAPKs) and cdc2-related kinases (CRKs) were identified as the major proteins specifically bound to the immobilized compound, suggesting their participation in the pharmacological effects of 2,4-diaminopyrimidines against trypanosomatid protozoan parasites. CONCLUSIONS/SIGNIFICANCE: Results show that 2,4-diaminopyrimidines have a good in vitro and in vivo pharmacological profile against trypanosomatid protozoans and that MAPKs and CRKs are potential molecular targets of these compounds. The 2,4-diminipyrimidines may serve as suitable leads for the development of novel treatments for HAT.

Discovery of novel orally bioavailable oxaborole 6-carboxamides that demonstrate cure in a murine model of late-stage central nervous system african trypanosomiasis.

We report the discovery of novel boron-containing molecules, exemplified by N-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-yl)-2-trifluoromethylbenzamide (AN3520) and 4-fluoro-N-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-yl)-2-trifluoromethylbenzamide (SCYX-6759), as potent compounds against Trypanosoma brucei in vitro, including the two subspecies responsible for human disease T. b. rhodesiense and T. b. gambiense. These oxaborole carboxamides cured stage 1 (hemolymphatic) trypanosomiasis infection in mice when administered orally at 2.5 to 10 mg/kg of body weight for 4 consecutive days. In stage 2 disease (central nervous system [CNS] involvement), mice infected with T. b. brucei were cured when AN3520 or SCYX-6759 were administered intraperitoneally or orally (50 mg/kg) twice daily for 7 days. Oxaborole-treated animals did not exhibit gross signs of compound-related acute or subchronic toxicity. Metabolism and pharmacokinetic studies in several species, including nonhuman primates, demonstrate that both SCYX-6759 and AN3520 are low-clearance compounds. Both compounds were well absorbed following oral dosing in multiple species and also demonstrated the ability to cross the blood-brain barrier with no evidence of interaction with the P-glycoprotein transporter. Overall, SCYX-6759 demonstrated superior pharmacokinetics, and this was reflected in better efficacy against stage 2 disease in the mouse model. On the whole, oxaboroles demonstrate potent activity against all T. brucei subspecies, excellent physicochemical profiles, in vitro metabolic stability, a low potential for CYP450 inhibition, a lack of active efflux by the P-glycoprotein transporter, and high permeability. These properties strongly suggest that these novel chemical entities are suitable leads for the development of new and effective orally administered treatments for human African trypanosomiasis.