Simple Field Assays to Check Quality of Current Artemisinin-Based Antimalarial Combination Formulations

INTRODUCTION: Malaria continues to be one of the major public health problems in Africa, Asia and Latin America. Artemisinin derivatives (ARTs; artesunate, artemether, and dihydroartemisinin) derived from the herb, Artemisia annua, are the most effective antimalarial drugs available providing rapid cures. The World Health Organisation (WHO) has recommended that all antimalarials must be combined with an artemisinin component (artemisinin-based combination therapy; ACT) for use as first line treatment against malaria. This class of drugs is now first-line policy in most malaria-endemic countries. Reports of ad hoc surveys from South East Asia show that up to 50% of the artesunate currently sold is counterfeit. Drug quality is rarely assessed in resource poor countries in part due to lack of dedicated laboratory facilities which are expensive to build, equip and maintain. With a view to address this unmet need we developed two novel colour reaction assays that can be used in the field to check the quality of ARTs. METHODS AND FINDINGS: Our assays utilise thin layer chromatography silica gel sheets and 2, 4 dinitrophenylhydrazine or 4-Benzoylamino-2, 5-dimethoxybenzenediazonium chloride hemi (zinc chloride) salt as the reagents showing a pink or blue product respectively only in the presence ARTs. We are able to detect as low as 10% of ARTs in ACTs (WINTHROP--artesunate/amodiaquine, Coartem--artemether/lumefantrine and Duocortexcin--dihydroartemisinin/piperaquine). The assays have been validated extensively by testing eighty readily accessible and widely used drugs in malaria endemic countries. None of the other antimalarial drugs or a range of commonly used excipients, antiretroviral drugs or other frequently used drugs from the WHO essential drugs list such as analgesics or antibiotics are detected with our assays. CONCLUSIONS: Our two independent assays requiring no specialist training are specific, simple to use, rapid, robust, reproducible, inexpensive and, have successfully resulted in detecting two counterfeit drugs within a small scale screening survey of over 100 declared artemisinin-containing drugs collected from various Asian and African countries. These promising results indicate that the assays will provide a useful first test to assure the quality of the ACTs formulations in resource poor malaria endemic areas when there is an absence of dedicated medicines quality laboratory facilities

Drug discovery and development for neglected diseases: the DNDi model

New models of drug discovery have been developed to overcome the lack of modern and effective drugs for neglected diseases such as human African trypanosomiasis (HAT; sleeping sickness), leishmaniasis, and Chagas disease, which have no financial viability for the pharmaceutical industry. With the purpose of combining the skills and research capacity in academia, pharmaceutical industry, and contract researchers, public–private partnerships or product development partnerships aim to create focused research consortia that address all aspects of drug discovery and development. These consortia not only emulate the projects within pharmaceutical and biotechnology industries, eg, identification and screening of libraries, medicinal chemistry, pharmacology and pharmacodynamics, formulation development, and manufacturing, but also use and strengthen existing capacity in disease-endemic countries, particularly for the conduct of clinical trials. The Drugs for Neglected Diseases initiative (DNDi) has adopted a model closely related to that of a virtual biotechnology company for the identification and optimization of drug leads. The application of this model to the development of drug candidates for the kinetoplastid infections of HAT, Chagas disease, and leishmaniasis has already led to the identification of new candidates issued from DNDi’s own discovery pipeline. This demonstrates that the model DNDi has been implementing is working but its DNDi, neglected diseases sustainability remains to be proven

Screening strategies to identify new chemical diversity for drug development to treat kinetoplastid infections

The Drugs for Neglected Diseases initiative (DNDi) has defined and implemented an early discovery strategy over the last few years, in fitting with its virtual R&D business model. This strategy relies on a medium- to high-throughput phenotypic assay platform to expedite the screening of compound libraries accessed through its collaborations with partners from the pharmaceutical industry. We review the pragmatic approaches used to select compound libraries for screening against kinetoplastids, taking into account screening capacity. The advantages, limitations and current achievements in identifying new quality series for further development into preclinical candidates are critically discussed, together with attractive new approaches currently under investigatio

Flavonoid profiling among wild type and related GM wheat varieties

Pleiotropic effects are one of the main concerns regarding genetically modified organisms (GMOs). This includes unintended side effects of the transgene or its genome insertion site on the regulation of other endogenous genes, which could potentially cause the accumulation of different secondary metabolites that may have not only an impact on diet as repeatedly worried by the public but also on the environment. Regarding amount and possible environmental effects, flavonoids represent the most prominent group of secondary metabolites in wheat. Many flavonoids function as signalling or defence molecules. We used a robust and reproducible analytical method to compare the flavonoid content of genetically modified (GM) wheat (Triticum aestivum L., Gramineae) expressing genes that confer increased fungal resistance with their non-GM siblings. The transgenes provide either a broad-spectrum fungal defence (chitinase/glucanase from barley) or bunt-specific resistance by a viral gene (KP4). Significant differences in flavonoid composition were found between different wheat varieties whereas different lines of GM wheat with increased antifungal resistance showed only minor differences in their flavonoid composition relative to their non-GM siblings. In a field test, no significant differences were detectable between infected and non-infected wheat of the same variety regardless of the presence of the transgene. Our results are in agreement with the hypothesis that the transgenes we used to increase wheat defence to fungal pathogens do not interfere with the flavonoid biosynthesis pathway. More significantly, the genetic background resulting from conventional breeding has a direct impact on the biological composition of flavonoids, and thus possibly on the environmen

Novel benzoxaborole, nitroimidazole and aminopyrazoles with activity against experimental cutaneous leishmaniasis.

OBJECTIVES: Drugs for Neglected Diseases initiative (DNDi) has identified three chemical lead series, the nitroimidazoles, benzoxaboroles and aminopyrazoles, as innovative treatments for visceral leishmaniasis. The leads discovered using phenotypic screening, were optimised following disease- and compound-specific criteria. Several leads of each series were progressed and preclinical drug candidates have been nominated. Here we evaluate the efficacy of the lead compounds of each of these three chemical classes in in vitro and in vivo models of cutaneous leishmaniasis. METHODS: The in vitro activity of fifty-five compounds was evaluated against the intracellular amastigotes of L. major, L. aethiopica, L. amazonensis, L. panamensis, L. mexicana and L. tropica. The drugs demonstrating potent activity (EC50 < 5 μM) against at least 4 of 6 species were subsequently evaluated in vivo in different L. major - BALB/c mouse models using a 5 or 10-day treatment with either the oral or topical formulations. Efficacy was expressed as lesion size (measured daily using callipers), parasite load (by quantitative PCR - DNA) and bioluminescence signal reduction relative to the untreated controls. RESULTS: The selected drug compounds (3 nitroimidazoles, 1 benzoxaborole and 3 aminopyrazoles) showed consistent and potent activity across a range of Leishmania species that are known to cause CL with EC50 values ranging from 0.29 to 18.3 μM. In all cases, this potent in vitro antileishmanial activity translated into high levels of efficacy with a linear dose-response against murine CL. When administered at 50 mg/kg/day, DNDI-0690 (nitroimidazole), DNDI-1047 (aminopyrazole) and DNDI-6148 (benzoxaborole) all resulted in a significant lesion size reduction (no visible nodule) and an approximate 2-log-fold reduction of the parasite load as measured by qPCR compared to the untreated control. CONCLUSIONS: The lead compounds DNDI-0690, DNDI-1047 and DNDI-6148 showed excellent activity across a range of Leishmania species in vitro and against L. major in mice. These compounds offer novel potential drugs for the treatment of CL

Addressing the most neglected diseases through an open research model: The discovery of fenarimols as novel drug candidates for eumycetoma

Eumycetoma is a chronic infectious disease characterized by a large subcutaneous mass, often caused by the fungus Madurella mycetomatis. A combination of surgery and prolonged medication is needed to treat this infection with a success rate of only 30%. There is, the

Novel 3-nitrotriazole-based amides and carbinols as bifunctional anti-Chagasic agents.

3-Nitro-1H-1,2,4-triazole-based amides with a linear, rigid core and 3-nitrotriazole-based fluconazole analogs were synthesized as dual functioning antitrypanosomal agents. Such compounds are excellent substrates for type I nitroreductase (NTR) located in the mitochondrion of trypanosomatids and, at the same time, act as inhibitors of the sterol 14α-demethylase (T. cruzi CYP51) enzyme. Because combination treatments against parasites are often superior to monotherapy, we believe that this emerging class of bifunctional compounds may introduce a new generation of antitrypanosomal drugs. In the present work, the synthesis and in vitro and in vivo evaluation of such compounds is discussed.This work was supported in part by internal funds of the Radiation Medicine Department at NorthShore University HealthSystem. Experiments on T. cruzi CYP51 were funded by NIH (GM067871, to G.I.L.). In vitro screenings against parasites were funded by DNDi. For that project, DNDi received funding from the following donors: Department for Internationl Development (DFID), U.K.; Bill & Melinda Gates Foundation (BMGF), USA; Reconstruction Credit Institution-Federal Ministry of Education and Research (KfW-BMBF), Germany; and Directorate-General for International Cooperation (DGIS), The Netherlands. B.A.-V. acknowledges financial support by FONDECYT Postdoctorado 3130364

Need for a Standardized Translational Drug Development Platform: Lessons Learned from the Repurposing of Drugs for COVID-19

In the absence of drugs to treat or prevent COVID-19, drug repurposing can be a valuable strategy. Despite a substantial number of clinical trials, drug repurposing did not deliver on its promise. While success was observed with some repurposed drugs (e.g., remdesivir, dexamethasone, tocilizumab, baricitinib), others failed to show clinical efficacy. One reason is the lack of clear translational processes based on adequate preclinical profiling before clinical evaluation. Combined with limitations of existing in vitro and in vivo models, there is a need for a systematic approach to urgent antiviral drug development in the context of a global pandemic. We implemented a methodology to test repurposed and experimental drugs to generate robust preclinical evidence for further clinical development. This translational drug development platform comprises in vitro, ex vivo, and in vivo models of SARS-CoV-2, along with pharmacokinetic modeling and simulation approaches to evaluate exposure levels in plasma and target organs. Here, we provide examples of identified repurposed antiviral drugs tested within our multidisciplinary collaboration to highlight lessons learned in urgent antiviral drug development during the COVID-19 pandemic. Our data confirm the importance of assessing in vitro and in vivo potency in multiple assays to boost the translatability of pre-clinical data. The value of pharmacokinetic modeling and simulations for compound prioritization is also discussed. We advocate the need for a standardized translational drug development platform for mild-to-moderate COVID-19 to generate preclinical evidence in support of clinical trials. We propose clear prerequisites for progression of drug candidates for repurposing into clinical trials. Further research is needed to gain a deeper understanding of the scope and limitations of the presented translational drug development platform

Antiprotozoal activity profiling of approved drugs : a starting point toward drug repositioning

Neglected tropical diseases cause significant morbidity and mortality and are a source of poverty in endemic countries. Only a few drugs are available to treat diseases such as leishmaniasis, Chagas' disease, human African trypanosomiasis and malaria. Since drug development is lengthy and expensive, a drug repurposing strategy offers an attractive fast-track approach to speed up the process. A set of 100 registered drugs with drug repositioning potential for neglected diseases was assembled and tested in vitro against four protozoan parasites associated with the aforementioned diseases. Several drugs and drug classes showed in vitro activity in those screening assays. The results are critically reviewed and discussed in the perspective of a follow-up drug repositioning strategy where R&D has to be addressed with limited resources