Analytical and mechanistic studies on the artemisinin antimalarials

Malaria is one of the most widespread of parasitic diseases. This disease is caused by protozoan parasites of Plasmodium, with P. falciparum and P. vivax being the most important. Artemisinin and its derivatives dihydroartemisinin (DHA), artesunate, artemether and arteether provide rapid cure. However, the drugs have short half-lives, and for this reason, and to suppress emergence of resistant parasite strains, the World Health Organization (WHO) has has implemented usage of artemisinin combination therapies (ACTs) such as dihydroartemisinin- piperaquine, artemether-lumefantrine, artesunate-mefloquine and artesunate-pyronaridine for the first line treatment of malaria. With the advent of artemisinins as clinical drugs, especially in the combination formulations, reliable analytical procedures for quantifying the amount of active pharmaceutical ingredient (API) that should be present in the formulated drug mixture have become crucial. Such procedures are also required to establish the nature of any degradation products that may appear during formulation or during storage of the formulated product. The first part of the thesis focusses on these aspects. The ACT consisting of DHA and piperaquine known as cotexcin is widely used, and DHA appears to decompose in such formulations. The identification of the degradant from DHA in cotexcin tablets from Thailand where cotexcin is used for treatment of non-severe malaria was carried out. Reverse phase HPLC, mass spectrometry and 1H NMR spectroscopy were used to identify this as the known tricarbonyl degradant from DHA. It was possible to quantify the amount of this degradant present in the tablet, and therefore establish how much of the DHA API was present. It was shown that the amount of DHA present was less than the specifications stipulated for use of the cotexcin ACT. Artemisone developed by collaboration between Bayer AG, Germany, and HKUST has passed clinical trials in combination with mefloquine. In the next phase of development, it is necessary to carry out protracted thermal stress testing of the formulated drug. Tablets of artemisone provided by the company engaged in carrying out this work were supplied to HKUST, and the degradants in the tablets were identified as known degradation products. A conclusion was then able to be drawn as to the suitability of artemisone in storage under thermally stressed conditions. It is a thermally more stable drug than DHA. Whenever a new drug appears on the market, fake or counterfeit drugs in which the API is replaced by a spurious, cheap substance that has no clinical worth rapidly appear. The practice is prominent in poorer countries where surveillance methods for testing authenticity are either non-existent or erratic, or subject to corruption. Up to 50% of artesunate currently sold in South East Asia is counterfeit, or substandard, and cases of fake artemisinin derivatives and ACTs have been reported from Africa. It is therefore crucial to develop a simple test suitable for use in the field to demonstrate authenticity of an artemisinin drug. Here, the aim was to evaluate a test based on the use of 2,4-dinitrophenylhydrazine in conjunction with a method originating at the London School of Hygiene and Tropical Medicine in London that is engaged in developing an analytical kit for use in the field. Artemisinins undergo a series of unzipping reactions to expose hydroperoxide and carbonyl groups, both of which may induce color changes by reaction with 2,4-dinitrophenylhydrazine to generate colored products. The reagent reacts with carbonyl compounds to produce a colored 2,4-dinitrophenylhydrazone. Here it was shown that it is possible to differentiate between artemisinins and a series of carbonyl compounds based on the colour of the products obtained. The underlying chemistry involving the reactions of the artemisinins and 2,4-dinitrophenylhydrazine was also clarified. Although the mechanism of action of action of just 15-20% of the drugs that appear in the World Drug Index is properly understood, that does not include the artemisinins. The need to understand mechanism of action is crucial, as the rational design of new drugs becomes easier, and toxicity, or of other side effects that may accompany use of a drug are better appreciated. How artemisinins with their peroxide pharmacophore may exert their effect has been subjected to an intense scrutiny. Many publications support the idea that ferrous iron, either in ferrous heme that is produced by breakdown of haemoglobin by the parasite, or of free ferrous iron liberated by metabolic breakdown of heme is the trigger for ‘activating’ artemisinins - the peroxide bond is cleaved according to Fenton chemistry to provide C-centred radicals that are the cytotoxic agents. However, there are problems with this hypothesis, not least of which is the nature of the target that the C-radicals are supposed to alkylate. The group at HKUST has shown the irrelevance of the Fenton reactions and the difficulty of inducing the artemisinins to react with ferrous iron under biomimetic conditions. Also the group has shown that artemisinins oxidize leucomethylene blue (LMB) to methylene blue (MB), and dihydroflavins such as the reduced conjugates dihydroriboflavin of riboflavin (RF), and FADH2 of the cofactor flavin adenine dinucleotide (FAD) to the corresponding flavins. At the same time, the artemisinins undergo two-electron reductions to the deoxygenated ether products. Artemisinins therefore may perturb redox balance in the malaria parasite by interfering with parasite flavoenzyme disulfide reductases such as glutathione reductase (GR) or thioredoxin reductase (TrxR), and may interfere with other flavoenzymes required for functioning of the malaria parasite. In this thesis, the examination of these reactions was continued by evaluating effects of reduced flavin cofactors and LMB with different artemisinins, and other peroxides that have antimalarial activities. The examinations were carried out by using the bacterial enzyme E. coli flavin reductase (Fre) in conjunction with NADPH to reduce quantitatively each of MB and flavins, including FAD to the reduced conjugates in aqueous buffer at pH 7.4, and then treating the solution with the antimalarial peroxides. In all cases, the reactions parallel those of artemisinin itself. The effect of artemisinins both on mammalian and parasite TrxR by examining effects on turnover of NADPH required for normal functioning of these enzymes in the presence of its substrate thioredoxin was evaluated; both enzymes are affected by artemisinins. Because artemisinins rapidly oxidize the reduced conjugates of MB and flavin cofactors, the effects of artemisinins on the reduced conjugates of other compounds that have similar redox properties to methylene blue have been evaluated. These included the widely-used anti-tuberculosis phenazine-type drug clofazimine, and the redox indicators thiazolyl blue tetrazolium bromide (MTT), Alamar blue and fluorescein that are used for establishing cell viabilities. As in the case of MB, the NADPH-Fre system was used to reduce quantitatively clofazimine, MTT and Alamar blue to their reduced conjugates. This is the first time that clofazimine, MTT and Alamar blue are shown to be substrates for Fre. It was shown that FADH2 generated from FAD and NADPH-Fre rapidly reduces clofazimine, MTT and Alamar blue. Therefore, in an intracellular environment, it is likely that reduced flavins actually initiate the redox cycling involving the important indicators MTT and Alamar blue – this has not been understood previously. Fluorescein was not a substrate for NADPH-Fre. All of the reduced conjugates were treated with the artemisinin. Except for the formazan reduced conjugate from MTT, the reduced conjugates were rapidly oxidized. These important findings indicate that artemisinin derivatives will be useful together with clofazimine and other similar redox-active drugs for treatment of tuberculosis. The results also indicate that caution has to be exercised in the use of Alamar blue as an end-point indicator for cytotoxicity where evaluation of cytotoxicity of artemisinins is carried out