Stability of the antimalarial drug dihydroartemisinin in under physiologically-relevant conditions : implications for clinical treatment, pharmacokinetic and in vitro assays

Artemisinins are peroxidic antimalarial drugs known to be very potent but chemically highly unstable; they degrade in the presence of ferrous iron, Fe(II)-heme or biological reductants. Less documented is how this translates into chemical stability and antimalarial activity across a range of conditions applying to in vitro testing and clinical situations. Dihydroartemisinin (DHA) is studied here because it is both an antimalarial drug on its own and the main metabolite of other artemisinins. The behavior of DHA in PBS, plasma or erythrocytes lysate at different temperatures and pH ranges was examined. The antimalarial activity of the residual drug was evaluated using the chemosensitivity assay on P. falciparum, and the extent of decomposition of DHA was established through use of HPLC-ECD analysis. The role of the Fe(II)-heme was investigated by blocking its reactivity using carbon monoxide. A significant reduction in the antimalarial activity of DHA was seen after incubation in plasma and to a lesser extent in erythrocytes lysate: activity was reduced by half after 3 hours and almost completely abolished after 24 hours. Serum-enriched media also affected DHA activity. Effects were temperature and pH-dependent and paralleled the increased rate of decomposition of DHA from pH 7 upwards and in plasma. These results suggest that particular care should be taken in conducting and interpreting in vitro studies, prone as they are to experimental and drug storage conditions. Disorders such as fever, hemolysis or acidosis associated with malaria severity may contribute to artemisinins instability and reduce their clinical efficacy

New hydrophilic riminophenazines as potent antiprotozoal agents

Malaria and leishmaniasis are life-threatening human parasitosis caused by protozoa-infected insect vectors. In most of affected countries, the expansive and hazardous therapies available to fight protozoan infections are generally harmed by the spread of drug resistance phenomena upon prolonged treatments. This scenario highlights the need of novel antiprotozoal agents hopefully able to act trough new mechanism(s) of action. Interestingly, the fat-soluble antimycobacterial drug Clofazimine was reported to exhibit a moderate antiprotozoal action and some interesting antileishmanial in vitro and in vivo effects were reported in few preliminary, yet promising, studies.1,4 Intrigued by these results, we have previously prepared a series of basic Clofazimine analogues which demonstrated the beneficial effects of the introduction of a basic head on the antiprotozoal activity.5 Here, to further investigate the role of balancing between the lipo- and hydrophilicity on the antiparasitic activity of these riminophenazines, we report the synthesis and the in vitro evaluation as leishmanicidal (L. tropica and L. infantum promastigotes) and antiplasmodial (chloroquine sensitive and resistant P. falciparum strains) agents of a family of hydrophilic C-2 aminopyridinyl substituted riminophenazines, bearing in C-3 differently decorated basic side chains. Results showed that most of the new compounds potently inhibited the growth of protozoa with IC50 in the high nanomolar range and underlined the key role of the hydrophilic C-2 aminopyridinyl moiety to improve the leishmanicidal activity. In addition, the length and the nature of the basic side chain differently influenced the antiprotozoal activity and the selectivity index versus mammalian cells, providing useful information for further structural optimizations

Oxidative inactivation of SARS-CoV-2 on photoactive AgNPs@Tio2 ceramic tiles

The current SARS-CoV-2 pandemic causes serious public health, social, and economic issues all over the globe. Surface transmission has been claimed as a possible SARS-CoV-2 infection route, especially in heavy contaminated environmental surfaces, including hospitals and crowded public places. Herein, we studied the deactivation of SARS-CoV-2 on photoactive AgNPs@TiO2 coated on industrial ceramic tiles under dark, UVA, and LED light irradiations. SARS-CoV-2 inactivation is effective under any light/dark conditions. The presence of AgNPs has an important key to limit the survival of SARS-CoV-2 in the dark; moreover, there is a synergistic action when TiO2 is decorated with Ag to enhance the virus photocatalytic inactivation even under LED. The radical oxidation was confirmed as the the central mechanism behind SARS-CoV-2 damage/inactivation by ESR analysis under LED light. Therefore, photoactive AgNPs@TiO2 ceramic tiles could be exploited to fight surface infections, especially during viral severe pandemics

In vitro multistage malaria transmission blocking activity of selected malaria box compounds

Purpose: Continuous efforts into the discovery and development of new antimalarials are required to face the emerging resistance of the parasite to available treatments. Thus, new effective drugs, ideally able to inhibit the Plasmodium life-cycle stages that cause the disease as well as those responsible for its transmission, are needed. Eight compounds from the Medicines for Malaria Venture (MMV) Malaria Box, potentially interfering with the parasite polyamine biosynthesis were selected and assessed in vitro for activity against malaria transmissible stages, namely mature gametocytes and early sporogonic stages. Methods: Compound activity against asexual blood stages of chloroquine-sensitive 3D7 and chloroquine-resistant W2 strains of Plasmodium falciparum was tested measuring the parasite lactate dehydrogenase activity. The gametocytocidal effect was determined against the P. falciparum 3D7elo1-pfs16-CBG99 strain with a luminescent method. The murine P. berghei CTRP.GFP strain was employed to assess compounds activities against early sporogonic stage development in an in vitro assay simulating mosquito midgut conditions. Results: Among the eight tested molecules, MMV000642, MMV000662 and MMV006429, containing a 1,2,3,4-tetrahydroisoquinoline-4-carboxamide chemical skeleton substituted at N-2, C-3 and C-4, displayed multi-stage activity. Activity against asexual blood stages of both strains was confirmed with values of IC50 (50% inhibitory concentration) in the range of 0.07\u20130.13 \ub5M. They were also active against mature stage V gametocytes with IC50 values below 5 \ub5M (range: 3.43\u20134.42 \ub5M). These molecules exhibited moderate effects on early sporogonic stage development, displaying IC50 values between 20 and 40 \ub5M. Conclusion: Given the multi-stage, transmission-blocking profiles of MMV000642, MMV000662, MMV006429, and their chemical characteristics, these compounds can be considered worthy for further optimisation toward a TCP5 or TCP6 target product profile proposed by MMV for transmission-blocking antimalarials

Target-Oriented Development Of Novel Antiprotozoal Agents: Celastrol Carboxamides As Inhibitors Of Leishmania Hsp90

The Leishmania isoform of the 90kDa Heat Shock Protein (LsHsp90), a chaperone known to assist the folding of more than 200 client proteins, was reported to be generally involved in parasite differentiation from promastigote to amastigote possessing a pivotal role during heat-induced cellular stress. Moreover, it was demonstrated that an impair of the native functions of LsHsp90 through the action of active-site inhibitors can exert a detrimental effect on the natural parasite life-cycle ultimately leading to its death. Celastrol is natural triterpene exhibiting a plethora of in vitro and in vivo activities. Among them, this pentacyclic compound is reported to possess a promising antiproliferative activity thanks to its ability of interacting with the chaperone cycle of the human isoform of Hsp90 (hHsp90). Moreover, celastrol derivatives (e.g. the methyl ester pristimerin, Figure 1) have also exhibited an interesting antiprotozoal activity. With the aim of building a target-oriented approach to treat Leishmania infections based on the inhibition of LsHsp90, we prepared two basic carboxamides celastrol derivatives (SS-1 and SS-2) to enhance its leishmanicidal activity and selectivity of action by deducting its unspecific cytotoxicity (measured as IC50 on HMEC-1 cell lines). Accordingly, celastrol and the two basic derivatives SS-1 and SS-2 were in vitro tested for their leishmanicidal activity against promastigotes of Leishmania tropica and L. infantum and, in parallel, their mechanism of action was investigated as well via ad hoc in vitro experiments using a recombinant Hsp90 from L. braziliensis (LbHsp90). In virtue of their pH sensitive basic heads, both SS-1 and SS-2 were found to be more potent (IC50 in the nanomolar range) and selective leishmanicidal agents than celastrol itself. Furthermore, we were able to demonstrate that SS-1 and SS-2 successfully (in vitro) inhibited the native kinase activity of LbHsp90 highlighting the key role of the inhibition of this chaperone in their mechanism of action

Benzimidazole derivatives endowed with potent antileishmanial activity

Two sets of benzimidazole derivatives were synthesised and tested in vitro for activity against promastigotes of Leishmania tropica and L. infantum. Most of the tested compounds resulted active against both Leishmania species, with IC50values in the low micromolar/sub-micromolar range. Among the set of 2-(long chain)alkyl benzimidazoles, whose heterocyclic head was quaternised, compound 8 resulted about 100-/200-fold more potent than miltefosine, even if the selectivity index (SI) versus HMEC-1 cells was only moderately improved. In the set of 2-benzyl and 2-phenyl benzimidazoles, bearing a basic side chain in position 1, compound 28 (2-(4-chlorobenzyl)-1-lupinyl-5-trifluoromethylbenzimidazole) was 12-/7-fold more potent than miltefosine, but exhibited a further improved SI. Therefore, compounds 8 and 28 represent interesting hit compounds, susceptible of structural modification to improve their safety profiles

Anti-plasmodial activity of Ailanthus excelsa

The anti-plasmodial activity of Ailanthus excelsa stem bark was investigated. The methanolic extract inhibited in vitro growth of chloroquine-sensitive (D10) and resistant strains (W2) of Plasmodium falciparum (IC50 4.6 and 2.8 \u3bcg/ml, respectively). The effect was retained in the chloroform fraction (3.1 and 2.1 \u3bcg/ml, respectively). The anti-plasmodial activity could be ascribed to the impairment of haemoglobin degradation through the inhibition of plasmepsin II activity (IC50 of 13.43 \ub1 1.74 \u3bcg/ml) and of the haem detoxification to haemozoin

Facile Preparation of N-Glycosylated 10-Piperazinyl Artemisinin Derivatives and Evaluation of Their Antimalarial and Cytotoxic Activities

According to the precepts that C-10 amino-artemisinins display optimum biological activities for the artemisinin drug class, and that attachment of a sugar enhances specificity of drug delivery, polarity and solubility so as to attenuate toxicity, we assessed the effects of attaching sugars to N-4 of the dihydroartemisinin (DHA)-piperazine derivative prepared in one step from DHA and piperazine. N-Glycosylated DHA-piperazine derivatives were obtained according to the Kotchetkov reaction by heating the DHA-piperazine with the sugar in a polar solvent. Structure of the D-glucose derivative is secured by X-ray crystallography. The D-galactose, L-rhamnose and D-xylose derivatives displayed IC50 values of 0.58\u20130.87 nM against different strains of Plasmodium falciparum (Pf ) and selectivity indices (SI) >195, on average, with respect to the mouse fibroblast WEHI-164 cell line. These activities are higher than those of the amino-artemisinin, artemisone (IC50 0.9\u20131.1 nM). Notably, the D-glucose, D-maltose and D-ribose derivatives were the most active against the myelogenous leukemia K562 cell line with IC50 values of 0.78\u20130.87 M and SI > 380 with respect to the human dermal fibroblasts (HDF). In comparison, artemisone has an IC50 of 0.26 M, and a SI of 88 with the same cell lines. Overall, the N-glycosylated DHA-piperazine derivatives display antimalarial activities that are greatly superior to O-glycosides previously obtained from DHA

High antiplasmodial activity of novel plasmepsins I and II inhibitors

The aim of this study was to develop new antiplasmodial compounds acting through distinct mechanisms during both the liver and the blood stages of the parasite life cycle. Compounds were designed on the basis of the "double-drug" approach: primaquine, which has been linked to statine-based inhibitors of plasmepsins (PLMs), the plasmodial aspartic proteases involved in degradation of hemeoglobin. The compounds were tested in vitro for anti-PLM I/PLM II activities and against chloroquine-sensitive (D10) and chloroquine-resistant (W2) strains of P. falciparum. An antiplasmodial activity (IC50) as low as 0.1 M was obtained, an excellent improvement in comparison with inhibitors previously reported (IC50 = 2-20 M). The killing activity was equally directed against both P. falciparum strains and was correlated to lipophilicity (calculated as ALogP), for all compounds but one (9). All compounds inhibited PLM I and PLM II in the nanomolar range (Ki = 1-700 nM). The most promising compounds (2, 6, 10) were not cytotoxic against human fibroblasts at 100 M and were highly selective for PLMs vs human cathepsin