Genotoxicity profile of fexinidazole—a drug candidate in clinical development for human African trypanomiasis (sleeping sickness)

The parasitic disease human African trypanomiasis (HAT), also known as sleeping sickness, is a highly neglected fatal condition endemic in sub-Saharan Africa, which is poorly treated with medicines that are toxic, no longer effective or very difficult to administer. New, safe, effective and easy-to-use treatments are urgently needed. Many nitroimidazoles possess antibacterial and antiprotozoal activity and examples such as tinidazole are used to treat trichomoniasis and guardiasis, but concerns about toxicity including genotoxicity limit their usefulness. Fexinidazole, a 2-substituted 5-nitroimidazole rediscovered by the Drugs for Neglected Diseases initiative (DNDi) after extensive compound mining of public and pharmaceutical company databases, has the potential to become a short-course, safe and effective oral treatment, curing both acute and chronic HAT. This paper describes the genotoxicity profile of fexinidazole and its two active metabolites, the sulfoxide and sulfone derivatives. All the three compounds are mutagenic in the Salmonella/Ames test; however, mutagenicity is either attenuated or lost in Ames Salmonella strains that lack one or more nitroreductase(s). It is known that these enzymes can nitroreduce compounds with low redox potentials, whereas their mammalian cell counterparts cannot, under normal conditions. Fexinidazole and its metabolites have low redox potentials and all mammalian cell assays to detect genetic toxicity, conducted for this study either in vitro (micronucleus test in human lymphocytes) or in vivo (ex vivo unscheduled DNA synthesis in rats; bone marrow micronucleus test in mice), were negative. Thus, fexinidazole does not pose a genotoxic hazard to patients and represents a promising drug candidate for HAT. Fexinidazole is expected to enter Phase II clinical trials in 201

Elevated ethyl methanesulfonate (EMS) in nelfinavir mesylate (Viracept®, Roche): overview

Roche's protease inhibitor nelfinavir mesylate (Viracept®) produced between March 2007-June 2007 was found to contain elevated levels of ethyl methanesulfonate (EMS), a known mutagen (alkylator) – leading to a global recall of the drug. EMS levels in a daily dose (2,500 mg Viracept/day) were predicted not to exceed a dose of ~2.75 mg/day (~0.055 mg/kg/day based on 50 kg patient). As existing toxicology data on EMS did not permit an adequate patient risk assessment, a comprehensive animal toxicology evaluation of EMS was conducted. General toxicity of EMS was investigated in rats over 28 days. Two studies for DNA damage were performed in mice; chromosomal damage was assessed using a micronucleus assay and gene mutations were detected using the MutaMouse transgenic model. In addition, experiments designed to extrapolate animal exposure to humans were undertaken. A general toxicity study showed that the toxicity of EMS occurred only at doses ≥ 60 mg/kg/day, which is far above that received by patients. Studies for chromosomal damage and mutations in mice demonstrated a clear threshold effect with EMS at 25 mg/kg/day, under chronic dosing conditions. Exposure analysis (Cmax) demonstrated that ~370-fold higher levels of EMS than that ingested by patients, are needed to saturate known, highly conserved, error-free, mammalian DNA repair mechanisms for alkylation. In summary, animal studies suggested that patients who took nelfinavir mesylate with elevated levels of EMS are at no increased risk for carcinogenicity or teratogenicity over their background risk, since mutations are prerequisites for such downstream events. These findings are potentially relevant to >40 marketed drugs that are mesylate salts

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

This article describes the preclinical profile of fexinidazole, a new drug candidate with the potential to become a novel, oral, safe and effective short-course treatment for curing both stage 1 and 2 human African trypanosomiasis and replace the old and highly problematic treatment modalities available today. Fexinidazole is orally available and rapidly metabolized in two metabolites having equivalent biological activity to the parent and contributing significantly to the in vivo efficacy in animal models of both stage 1 and 2 HAT. Animal toxicology studies indicate that fexinidazole has an excellent safety profile, with no particular issues identified. Fexinidazole is a 5-nitroimidazole and, whilst it is Ames-positive, it is devoid of any genetic toxicity in mammalian cells and therefore does not pose a genotoxic risk for use in man. Fexinidazole, which was rediscovered through a process of compound mining, is the first new drug candidate for stage 2 HAT having entered clinical trials in thirty years, and has the potential to revolutionize therapy of this fatal disease at a cost that is acceptable in the endemic regions

How to Reduce False Positive Results When Undertaking In Vitro Genotoxicity Testing and Thus Avoid Unnecessary Follow-up Animal Tests: Report of an ECVAM Workshop

Workshop participants agreed that genotoxicity tests in mammalian cells in vitro produce a remarkably high and unacceptable occurrence of irrelevant positive results (e.g. when compared with rodent carcinogenicity). As reported in several recent reviews, the rate of irrelevant positives (i.e. low specificity) for some studies using in vitro methods (when compared to this "gold standard") means that an increased number of test articles are subjected to additional in vivo genotoxicity testing, in many cases before e.g. the efficacy (in the case of pharmaceuticals) of the compound has been evaluated. If in vitro tests were more predictive for in vivo genotoxicity and carcinogenicity (i.e. fewer false positives) then there would be a significant reduction in the number of animals used. Beyond animal (or human) carcinogenicity as the "gold standard", it is acknowledged that genotoxicity tests provide much information about cellular behaviour, cell division processes and cellular fate to a (geno)toxic insult. Since the disease impact of these effects is seldomly known, and a verification of relevant toxicity is normally also the subject of (sub)chronic animal studies, the prediction of in vivo relevant results from in vitro genotoxicity tests is also important for aspects that may not have a direct impact on carcinogenesis as the ultimate endpoint of concern. In order to address the high rate of in vitro false positive results, a 2-day workshop was held at the European Centre for the Validation of Alternative Methods (ECVAM), Ispra, Italy in April 2006. More than 20 genotoxicity experts from academia, government and industry were invited to review data from the currently available cell systems, to discuss whether there exist cells and test systems that have a reduced tendency to false positive results, to review potential modifications to existing protocols and cell systems that might result in improved specificity, and to review the performance of some new test systems that show promise of improved specificity without sacrificing sensitivity.JRC.I.2-Validation of biomedical testing method