Marcel Kaiser

Antitrypanosomal and Antileishmanial Activities of Flavonoids and Their Analogues: In Vitro, In Vivo, Structure-Activity Relationship, and Quantitative Structure-Activity Relationship Studies

ABSTRACT Trypanosomiasis and leishmaniasis are important parasitic diseases affecting millions of people in Africa, Asia, and South America. In a previous study, we identified several flavonoid glycosides as antiprotozoal principles from a Turkish plant. Here we surveyed a large set of flavonoid aglycones and glycosides, as well as a panel of other related compounds of phenolic and phenylpropanoid nature, for their in vitro activities against Trypanosoma brucei rhodesiense, Trypanosoma cruzi, and Leishmania donovani. The cytotoxicities of more than 100 compounds for mammalian L6 cells were also assessed and compared to their antiparasitic activities. Several compounds were investigated in vivo for their antileishmanial and antitrypanosomal efficacies in mouse models. Overall, the best in vitro trypanocidal activity for T. brucei rhodesiense was exerted by 7,8-dihydroxyflavone (50% inhibitory concentration [IC50], 68 ng/ml), followed by 3-hydroxyflavone, rhamnetin, and 7,8,3′,4′-tetrahydroxyflavone (IC50s, 0.5 μg/ml) and catechol (IC50, 0.8 μg/ml). The activity against T. cruzi was moderate, and only chrysin dimethylether and 3-hydroxydaidzein had IC50s less than 5.0 μg/ml. The majority of the metabolites tested possessed remarkable leishmanicidal potential. Fisetin, 3-hydroxyflavone, luteolin, and quercetin were the most potent, giving IC50s of 0.6, 0.7, 0.8, and 1.0 μg/ml, respectively. 7,8-Dihydroxyflavone and quercetin appeared to ameliorate parasitic infections in mouse models. Generally, the test compounds lacked cytotoxicity in vitro and in vivo. By screening a large number of flavonoids and analogues, we were able to establish some general trends with respect to the structure-activity relationship, but it was not possible to draw clear and detailed quantitative structure-activity relationships for any of the bioactivities by two different approaches. However, our results can help in directing the rational design of 7,8-dihydroxyflavone and quercetin derivatives as potent and effective antiprotozoal agents.

Fexinidazole – A New Oral Nitroimidazole Drug Candidate Entering Clinical Development for the Treatment of Sleeping Sickness

Background Human African trypanosomiasis (HAT), also known as sleeping sickness, is a fatal parasitic disease caused by trypanosomes. Current treatment options for HAT are scarce, toxic, no longer effective, or very difficult to administer, in particular for the advanced, fatal stage of the disease (stage 2, chronic HAT). New safe, effective and easy-to-use treatments are urgently needed. Here it is shown that fexinidazole, a 2-substituted 5-nitroimidazole rediscovered by the Drugs for Neglected Diseases initiative (DNDi) after extensive compound mining efforts of more than 700 new and existing nitroheterocycles, could be a short-course, safe and effective oral treatment curing both acute and chronic HAT and that could be implemented at the primary health care level. To complete the preclinical development and meet the regulatory requirements before initiating human trials, the anti-parasitic properties and the pharmacokinetic, metabolic and toxicological profile of fexinidazole have been assessed. Methods and Findings Standard in vitro and in vivo anti-parasitic activity assays were conducted to assess drug efficacy in experimental models for HAT. In parallel, a full range of preclinical pharmacology and safety studies, as required by international regulatory guidelines before initiating human studies, have been conducted. Fexinidazole is moderately active in vitro against African trypanosomes (IC50 against laboratory strains and recent clinical isolates ranged between 0.16 and 0.93 µg/mL) and oral administration of fexinidazole at doses of 100 mg/kg/day for 4 days or 200 mg/kg/day for 5 days cured mice with acute and chronic infection respectively, the latter being a model for the advanced and fatal stage of the disease when parasites have disseminated into the brain. In laboratory animals, fexinidazole is well absorbed after oral administration and readily distributes throughout the body, including the brain. The absolute bioavailability of oral fexinidazole was 41% in mice, 30% in rats, and 10% in dogs. Furthermore, fexinidazole is rapidly metabolised in vivo to at least two biologically active metabolites (a sulfoxide and a sulfone derivative) that likely account for a significant portion of the therapeutic effect. Key pharmacokinetic parameter after oral absorption in mice for fexinidazole and its sulfoxide and sulfone metabolites are a Cmax of 500, 14171 and 13651 ng/mL respectively, and an AUC0–24 of 424, 45031 and 96286 h.ng/mL respectively. Essentially similar PK profiles were observed in rats and dogs. Toxicology studies (including safety pharmacology and 4-weeks repeated-dose toxicokinetics in rat and dog) have shown that fexinidazole is well tolerated. The No Observed Adverse Event Levels in the 4-weeks repeated dose toxicity studies in rats and dogs was 200 mg/kg/day in both species, with no issues of concern identified for doses up to 800 mg/kg/day. While fexinidazole, like many nitroheterocycles, is mutagenic in the Ames test due to bacterial specific metabolism, it is not genotoxic to mammalian cells in vitro or in vivo as assessed in an in vitro micronucleus test on human lymphocytes, an in vivo mouse bone marrow micronucleus test, and an ex vivo unscheduled DNA synthesis test in rats. Conclusions The results of the preclinical pharmacological and safety studies indicate that fexinidazole is a safe and effective oral drug candidate with no untoward effects that would preclude evaluation in man. The drug has entered first-in-human phase I studies in September 2009. Fexinidazole is the first new clinical drug candidate with the potential for treating advanced-stage sleeping sickness in thirty years.

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.

Inhibitory Activity of Marine Sponge-Derived Natural Products against Parasitic Protozoa

In this study, thirteen sponge-derived terpenoids, including five linear furanoterpenes: furospinulosin-1 (1), furospinulosin-2 (2), furospongin-1 (3), furospongin-4 (4), and demethylfurospongin-4 (5); four linear meroterpenes: 2-(hexaprenylmethyl)-2-methylchromenol (6), 4-hydroxy-3-octaprenylbenzoic acid (7), 4-hydroxy-3-tetraprenyl-phenylacetic acid (8), and heptaprenyl-p-quinol (9); a linear triterpene, squalene (10); two spongian-type diterpenes dorisenone D (11) and 11β-acetoxyspongi-12-en-16-one (12); a scalarane-type sesterterpene; 12-epi-deoxoscalarin (13), as well as an indole alkaloid, tryptophol (14) were screened for their in vitro activity against four parasitic protozoa; Trypanosoma brucei rhodesiense, Trypanosoma cruzi, Leishmania donovani and Plasmodium falciparum. Cytotoxic potential of the compounds on mammalian cells was also assessed. All compounds were active against T. brucei rhodesiense, with compound 8 being the most potent (IC50 0.60 μg/mL), whereas 9 and 12 were the most active compounds against T. cruzi, with IC50 values around 4 μg/mL. Compound 12 showed the strongest leishmanicidal activity (IC50 0.75 μg/mL), which was comparable to that of miltefosine (IC50 0.20 μg/mL). The best antiplasmodial effect was exerted by compound 11 (IC50 0.43 μg/mL), followed by compounds 7, 10, and 12 with IC50 values around 1 μg/mL. Compounds 9, 11 and 12 exhibited, besides their antiprotozoal activity, also some cytotoxicity, whereas all other compounds had low or no cytotoxicity towards the mammalian cell line. This is the first report of antiprotozoal activity of marine metabolites 1–14, and points out the potential of marine sponges in discovery of new antiprotozoal lead compounds.

Quantitative Structure ‒ Antiprotozoal Activity Relationships of Sesquiterpene Lactones

Prompted by results of our previous studies where we found high activity of some sesquiterpene lactones (STLs) against Trypanosoma brucei rhodesiense (which causes East African sleeping sickness), we have now conducted a structure-(in-vitro)-activity study on a set of 40 STLs against T. brucei rhodesiense, T. cruzi, Leishmania donovani and Plasmodium falciparum. Furthermore, cytotoxic activity against L6 rat skeletal myoblast cells was assessed. Some of the compounds possess high activity, especially against T. brucei (e.g. helenalin and some of its esters with IC50-values of 0.05-0.1 µM, which is about 10 times lower than their cytotoxic activity). It was found that all investigated antiprotozoal activities are significantly correlated with cytotoxicity and the major determinants for activity are α,β-unsaturated structural elements, also known to be essential for other biological activities of STLs. It was observed, however, that certain compounds are considerably more toxic against protozoa than against mammalian cells while others are more cytotoxic than active against the protozoa. A comparative QSAR analysis was therefore undertaken, in order to discern the antiparasitic activity of STLs against T. brucei and cytotoxicity. Both activities were found to depend to a large extent on the same structural elements and molecular properties. The observed variance in the biological data can be explained in terms of subtle variations in the relative influences of various molecular descriptors.

Antitrypanosomal Activity of Fexinidazole, a New Oral Nitroimidazole Drug Candidate for Treatment of Sleeping Sickness

ABSTRACT Fexinidazole is a 5-nitroimidazole drug currently in clinical development for the treatment of human sleeping sickness (human African trypanosomiasis [HAT]), caused by infection with species of the protozoan parasite Trypanosoma brucei. The compound and its two principal metabolites, sulfoxide and sulfone, have been assessed for their ability to kill a range of T. brucei parasite strains in vitro and to cure both acute and chronic HAT disease models in the mouse. The parent molecule and both metabolites have shown trypanocidal activity in vitro in the 0.7-to-3.3 μM (0.2-to-0.9 μg/ml) range against all parasite strains tested. In vivo, fexinidazole is orally effective in curing both acute and chronic diseases in the mouse at doses of 100 mg/kg of body weight/day for 4 days and 200 mg/kg/day for 5 days, respectively. Pharmacokinetic data indicate that it is likely that the sulfoxide and sulfone metabolites provide most, if not all, of the in vivo killing activity. Fexinidazole and its metabolites require up to 48 h exposure in order to induce maximal trypanocidal efficacy in vitro. The parent drug and its metabolites show no in vitro cross-reactivity in terms of trypanocidal activity with either themselves or other known trypanocidal drugs in use in humans. The in vitro and in vivo antitrypanosomal activities of fexinidazole and its two principal metabolites provide evidence that the compound has the potential to be an effective oral treatment for both the T. b. gambiense and T. b. rhodesiense forms of human sleeping sickness and both stages of the disease.

Antiparasitic agents : new drugs on the horizon

The need for new drugs against tropical parasites such as Plasmodium falciparum and Trypanosoma brucei is persistent since problems with resistance and toxicity are jeopardizing the currently available medicines. Public-private partnerships aiming to develop new medicines for malaria and sleeping sickness have, over the past 12 years, brought forward several drug candidates that have entered clinical trials. These are the synthetic peroxide OZ439 and the spiroindolone NITD609 against P. falciparum, fexinidazole and the oxaborole SCYX-7158 against T. brucei. A further class of high chemotherapeutic potential are the diamidines, novel members of which may serve as back-up compounds against trypanosomes and other parasites. Thus, finally, new therapeutic agents against malaria and sleeping sickness are within reach.

New bis(2-aminoimidazoline) and bisguanidine DNA minor groove binders with potent in vivo antitrypanosomal and antiplasmodial activity.

A series of 75 guanidine and 2-aminoimidazoline analogue molecules were assayed in vitro against Trypanosoma brucei rhodesiense STIB900 and Plasmodium falciparum K1. The dicationic diphenyl compounds exhibited the best activities with IC 50 values against T. b. rhodesiense and P. falciparum in the nanomolar range. Five compounds (7b, 9a, 9b, 10b, and 14b) cured 100% of treated mice upon ip administration at 20 mg/kg in the difficult to cure T. b. rhodesiense STIB900 mouse model. Overall, the compounds that bear the 2-aminoimidazoline cations benefit from better safety profiles than the guanidine counterparts. The observation of a correlation between DNA binding affinity at AT sites and trypanocidal activity for three series of compounds supported the view of a mechanism of antitrypanosomal action due in part to the formation of a DNA complex. No correlation between antiplasmodial activity and in vitro inhibition of ferriprotoporphyrin IX biomineralisation was observed, suggesting that additional mechanism of action is likely to be involved.

DMSO‐Mediated Ligand Dissociation: Renaissance for Biological Activity of N‐Heterocyclic‐[Ru(η6‐arene)Cl2] Drug Candidates

Slipped under the radar? (1) H NMR spectroscopic examination revealed that [Ru(η(6) -arene)Cl2 (L)] (L=N-heterocyclic ligands) complexes readily undergo ligand exchange reaction in DMSO, a popular medium for preparing stock solutions for biological screening. It is therefore highly important for researchers to study stability in DMSO before reporting on the biological activity of such type of complexes.

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.