Nigel Yarlett

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 Cmax >10 µg/mL and AUC0–24 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.

Inducible Resistance to Oxidant Stress in the Protozoan Leishmania chagasi

Leishmania sp. protozoa are introduced into a mammalian skin by a sandfly vector, whereupon they encounter increased temperature and toxic oxidants generated during phagocytosis. We studied the effects of 37 °C “heat shock” or sublethal menadione, which generates superoxide and hydrogen peroxide, on Leishmania chagasi virulence. Both heat and menadione caused parasites to become more resistant to H2O2-mediated toxicity. Peroxide resistance was also induced as promastigotes developed in culture from logarithmic to their virulent stationary phase form. Peroxide resistance was not associated with an increase in reduced thiols (trypanothione and glutathione) or increased activity of ornithine decarboxylase, which is rate-limiting in trypanothione synthesis. Membrane lipophosphoglycan increased in size as parasites developed to stationary phase but not after environmental exposures. Instead, parasites underwent a heat shock response upon exposure to heat or sublethal menadione, detected by increased levels of HSP70. Transfection of promastigotes withL. chagasi HSP70 caused a heat-inducible increase in resistance to peroxide, implying it is involved in antioxidant defense. We conclude that leishmania have redundant mechanisms for resisting toxic oxidants. Some are induced during developmental change and others are induced in response to environmental stress.

Antioxidant defences in the microaerophilic protozoan Trichomonas vaginalis : comparison of metronidazole-resistant and sensitive strains

The sensitivity of the microaerophilic protozoan Trichomonas vaginalis to oxygen and products of its reduction, and the antioxidant defences employed by this organism, were investigated. Studies revealed that this amitochondrial flagellate is sensitive to oxygen tensions above those experienced in situ in the vagina (i.e. > 60 microM) and that metronidazole-resistant strains (CDC 85 and IR78) were more sensitive to elevated oxygen levels than a metronidazole-sensitive isolate (1910). In the presence of radical scavengers, inactivation of organisms at 60 microM oxygen was significantly lessened. Investigation of the antioxidant enzymes present in this organism revealed that activities of peroxide-reducing enzymes (e.g. catalase and general peroxidase) were not detectable, but that a cyanide-insensitive, azide-sensitive superoxide dismutase was present in cell extracts. Measurement of thiol-cycling enzymes indicated that NADPH could drive the reduction of oxidized glutathione (thiol reductase); however, the corresponding peroxidase activity was not detected. Analysis of thiols in whole cells of T. vaginalis indicated that glutathione was absent, but high levels of other thiols, propanethiol, methanethiol and H2S, were present. No significant differences were detected in thiol levels or antioxidant enzyme activities on comparison of metronidazole-sensitive and resistant strains. These results indicate that the sensitivity of T. vaginalis to oxygen above physiological levels is due to the lack of adequate peroxide-reducing enzymes and radical-scavenging mechanisms.

Discovery of Novel Orally Bioavailable Oxaborole 6-Carboxamides That Demonstrate Cure in a Murine Model of Late-Stage Central Nervous System African Trypanosomiasis

ABSTRACT 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.

Polyamine biosynthesis in Cryptosporidium parvum and its implications for chemotherapy

This study demonstrates that polyamine biosynthesis in Cryptosporidium parvum occurs via a pathway chiefly found in plants and some bacteria. The lead enzyme of this pathway, arginine decarboxylase (ADC) was sensitive to the specific, irreversible inhibitor DL-alpha-difluoromethyl-arginine (IC50 30 microM), and intracellular growth of C. parvum was significantly reduced by inhibitors of ADC. No activity was detected using ornithine as substrate, and the irreversible inhibitor of ornithine decarboxylase, DL-alpha-difluoromethyl-ornithine, had no effect upon ADC activity or upon growth of the parasite. Back-conversion of spermine to spermidine and putrescine via spermidine:spermine-N1-acetyltransferase (SSAT) was also detected. Compounds such as his(ethyl)norspermine, which have been demonstrated to down-regulate SSAT activity in tumor cells, were synergistic in the inhibition of growth when used in combination with inhibitors of the forward pathway. Thus, C. parvum differs fundamentally in its polyamine metabolism from the majority of eukaryotes, including humans. Such differences indicate that polyamine metabolism may serve as a chemotherapeutic target in this organism.

Effect of dl-α-difluoromethylornithine on methionine cycle intermediates in Trypanosoma brucei brucei

Activities of enzymes involved in transmethylation reactions were determined in bloodstream trypomastigotes of Trypanosoma brucei brucei infection in rats. S-Adenosyl-l-methionine synthetase (EC 2.5.1.6), S-adenosyl-l-homocysteine hydrolase (EC 3.3.1.1), cystathionine synthase (EC 4.2.1.21), as well as several transmethylases were detected and localized in cytosolic rather than particulate fractions. High performance liquid chromatography analysis of methionine cycle intermediates in cells from untreated rats and from rats treated with the ornithine decarboxylase inhibitor dl-α-difluoromethylornithine (DFMO) indicated that the inhibitor causes pronounced changes in concentrations of these intermediates and dramatically alters the methylation index of the cell. These findings demonstrate another in the wide range of metabolite disturbances attributable to DFMO and reflect the belief that multiple biochemical events are a sequel of its action on trypanosomes.

Novel Synthetic Polyamines Are Effective in the Treatment of Experimental Microsporidiosis, an Opportunistic AIDS-Associated Infection

ABSTRACT Microsporidia are eukaryotic obligate intracellular protists that are emerging pathogens in immunocompromised hosts, such as patients with AIDS or patients who have undergone organ transplantation. We have demonstrated in vitro and in vivo that synthetic polyamine analogs are effective antimicrosporidial agents with a broad therapeutic window. CD8-knockout mice or nude mice infected with the microsporidian Encephalitozoon cuniculi were cured when they were treated with four different novel polyamine analogs at doses ranging from 1.25 to 5 mg/kg of body weight/day for a total of 10 days. Cured animals demonstrated no evidence of parasitemia by either PCR or histologic staining of tissues 30 days after untreated control animals died.

Discovery of Novel Benzoxaborole-Based Potent Antitrypanosomal Agents

We report the discovery of benzoxaborole antitrypanosomal agents and their structure-activity relationships on central linkage groups and different substitution patterns in the sulfur-linked series. The compounds showed in vitro growth inhibition IC50 values as low as 0.02 μg/mL and in vivo efficacy in acute murine infection models against Tryapnosoma brucei.

Resistance to dl-α-difluoromethylornithine by clinical isolates of Trypanosoma brucei rhodesiense: Role of S-adenosylmethionine

The ornithine decarboxylase (ODC) inhibitor DL-alpha-difluoromethylornithine (DFMO) has emerged as a new treatment for West African sleeping sickness but is less effective against East African sleeping sickness. We examined uncloned clinical isolates of Trypanosoma brucei rhodesiense, agent of the disease in East Africa, which were refractory to DFMO in laboratory infections, for characteristics that would explain their resistance. None of the isolates were from patients treated with DFMO. Two isolates took up [3H]DFMO at 50-70% lower rates than drug-sensitive strains but ODC activities, Ki values for DFMO, spermidine and spermine uptake rates, polyamine content and inhibition of polyamine metabolism by DFMO were statistically (P < 0.05) similar between sensitive and refractory isolates. One cloned strain, continuously passaged in vivo under DFMO pressure and included for comparison, had > 85% lower ODC activity and up to 14-fold higher putrescine uptake rates than sensitive controls. A statistically important trend was the metabolism of S-adenosylmethionine (AdoMet): activities of AdoMet synthetase and AdoMet decarboxylase were 2- to 5-fold and 3- to 40-fold lower in resistant strains, respectively, while intracellular AdoMet pools (AdoMet + decarboxylated AdoMet) that were > 60-fold elevated in sensitive strains during DFMO treatment, increased only 9-fold in refractory isolates. The extreme elevation of the AdoMet pool in sensitive isolates from 0.7 to 44 nmol/mg protein and an intracellular pool concentration of approximately 5 mM may lead to an imbalance in methylation of proteins or other cell constituents as a consequence of DFMO action. These studies indicate that the metabolism of AdoMet is altered significantly in DFMO refractory isolates and suggest that differences in AdoMet metabolism may be responsible for increased tolerance to DFMO.

Oxygen Affinities of the Hydrogenosome-containing Protozoa Tritrichomonas foetus and Dasytricha ruminantium, and Two Aerobic Protozoa, Determined by Bacterial Bioluminescence

Oxygen-dependent bioluminescence of Photobacterium (Vibrio) fischeri was used to measure oxygen affinities of four protozoa. The aerobic organisms Acanthamoeba castellanii and Tetrahymena pyriformis showed apparent Km values for O2 of 0.42 and 2.43 microM respectively. The aerotolerant anaerobe Tritrichomonas foetus, and the more strictly anaerobic rumen ciliate Dasytricha ruminantium, both of which have hydrogenosomes, respired with apparent Km values of 1.08 and 1.70 microM-O2. We conclude that mitochondrial respiration is not the only process conferring on organisms a high affinity for O2.