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

Synthesis and antiplasmodial activity of novel bioinspired imidazolidinedione derivatives

Malaria is an enormous threat to public health, due to the emergence of Plasmodium fal-ciparum resistance to widely-used antimalarials, such as chloroquine (CQ). Current antimalarial drugs are aromatic heterocyclic derivatives, most often containing a basic component with an added alkyl chain in their chemical structure. While these drugs are effective, they have many side effects. This paper presents the synthesis and preliminary physicochemical characterisation of novel bioinspired imidazolidinedione derivatives, where the imidazolidinedione core was linked via the alkylene chain and the basic piperazine component to the bicyclic system. These compounds were tested against the asexual stages of two strains of P. falciparum—the chloro-quine-sensitive (D10) and chloroquine-resistant (W2) strains. In parallel, in vitro cytotoxicity was investigated on a human keratinocyte cell line, as well as their hemolytic activity. The results demonstrated that the antiplasmodial effects were stronger against the W2 strain (IC50 between 2424.15–5648.07 ng/mL (4.98–11.95 μM)), compared to the D10 strain (6202.00–9659.70 ng/mL (12.75–19.85 μM)). These molecules were also non-hemolytic to human erythrocytes at a concen-tration active towards the parasite, but with low toxicity to mammalian cell line. The synthetized derivatives, possessing enhanced antimalarial activity against the CQ-resistant strain of P. falcipa-rum, appear to be interesting antimalarial drug candidates

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

In Vitro Antimalarial Activity of Inhibitors of the Human GTPase Rac1

Malaria accounts for millions of cases and thousands of deaths every year. In the absence of an effective vaccine, drugs are still the most important tool in the fight against the disease. Plasmodium parasites developed resistance to all classes of known antimalarial drugs. Thus, the search for antimalarial drugs with novel mechanisms of action is compelling. The human GTPase Rac1 plays a role in parasite invasion of the host cell in many intracellular pathogens. Also, in Plasmodium falciparum, the involvement of Rac1 during both the invasion process and parasite intracellular development was suggested. The aim of this work is to test a panel of Rac1 inhibitors as potential antimalarial drugs. Fourteen commercially available or newly synthesized inhibitors of Rac1 were tested for antimalarial activity. Among these, EHop-016 was the most effective against P. falciparum in vitro, with nanomolar 50% inhibitory concentrations (IC50s) (138.8 ± 16.0 nM on the chloroquine-sensitive D10 strain and 321.5 ± 28.5 nM on the chloroquine-resistant W2 strain) and a selectivity index of 37.8. EHop-016 did not inhibit parasite invasion of red blood cells but affected parasite growth inside them. Among the tested Rac1 inhibitors, EHop-016 showed promising activity that raises attention to this class of molecules as potential antimalarials and deserves further investigation

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

Antiprotozoal activity of novel diaryliminophenazines

Recently, we synthesized a set of novel iminofenazines bearing a bicyclic basic head linked through an alkyl chain to the imino nitrogen in position 3 on the phenazine nucleus (Fig.1). Most of these compounds inhibited the growth of different species of Leishmania promastigotes as well as of chloroquine sensitive (CQ-S) and chloroquine resistant (CQ-R) strains of P. falciparum with IC50 in the submicromolar range. Unfortunately, these compounds exhibited also a significant toxicity against the human endothelial cell line HMEC-1 with IC50 in the low micromolar range and with a consequent low selectivity index. Figure1.Structures of the previously synthesized compounds. To continue the studies on the antiprotozoal potentialities of this class of compounds and with the aim to improve their activity and selectivity on protozoa, we have now synthesized novel compounds characterized by the replacement of the aniline moiety in pos. 2 of the phenazine nucleus with an aminopyridine, and/or by a quaternarization of the basic nitrogen in the side chain with a methyl group (Fig.2). Figure 2. Structures of the new compounds synthesized. The in vitro activity of the new compounds on Leishmania promastigotes and on CQ-S and CQ-R strains of P. falciparum, as well as on the HMEC-1 cell line will be presented and discussed. References [1] A. Barteselli, M. Gavazzi, N. Basilico, S. Parapini, D. Taramelli, A. Sparatore. Clofazimine analogs with antileishmanial and antimalarial activities. XXII National Meeting on Medicinal Chemistry, Roma 2013

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

Role of Stereochemistry on the Biological Activity of Nature-Inspired 3-Br-Acivicin Isomers and Derivatives

Chiral natural compounds are often biosynthesized in an enantiomerically pure fashion, and stereochemistry plays a pivotal role in biological activity. Herein, we investigated the significance of chirality for nature-inspired 3-Br-acivicin (3-BA) and its derivatives. The three unnatural isomers of 3-BA and its ester and amide derivatives were prepared and characterized for their antimalarial activity. Only the (5S, αS) isomers displayed significant antiplasmodial activity, revealing that their uptake might be mediated by the L-amino acid transport system, which is known to mediate the acivicin membrane’s permeability. In addition, we investigated the inhibitory activity towards Plasmodium falciparum glyceraldehyde 3-phosphate dehydrogenase (PfGAPDH) since it is involved in the multitarget mechanism of action of 3-BA. Molecular modeling has shed light on the structural and stereochemical requirements for an efficient interaction with PfGAPDH, leading to covalent irreversible binding and enzyme inactivation. While stereochemistry affects the target binding only for two subclasses (1a–d and 4a–d), it leads to significant differences in the antimalarial activity for all subclasses, suggesting that a stereoselective uptake might be responsible for the enhanced biological activity of the (5S, αS) isomers

Multiplex array analysis of circulating cytokines and chemokines in COVID-19 patients during the first wave of the SARS-CoV-2 pandemic in Milan, Italy.

Background: The systemic inflammatory syndrome called "cytokine storm" has been described in COVID-19 pathogenesis, contributing to disease severity. The analysis of cytokine and chemokine levels in the blood of 21 SARS-CoV-2 positive patients throughout the phases of the pandemic has been studied to understand immune response dysregulation and identify potential disease biomarkers for new treatments. The present work reports the cytokine and chemokine levels in sera from a small cohort of individuals primarily infected with SARS-CoV-2 during the first wave of the COVID-19 pandemic in Milan (Italy). Results: Among the 27 cytokines and chemokines investigated, a significant higher expression of Interleukin-9 (IL-9), IP-10 (CXCL10), MCP-1 (CCL2) and RANTES (CCL-5) in infected patients compared to uninfected subjects was observed. When the change in cytokine/chemokine levels was monitored over time, from the hospitalization day to discharge, only IL-6 and IP-10 showed a significant decrease. Consistent with these findings, a significant negative correlation was observed between IP-10 and anti-Spike IgG antibodies in infected individuals. In contrast, IL-17 was positively correlated with the production of IgG against SARS-CoV-2. Conclusions: The cytokine storm and the modulation of cytokine levels by SARS-CoV-2 infection are hallmarks of COVID-19. The current global immunity profile largely stems from widespread vaccination campaigns and previous infection exposures. Consequently, the immunological features and dynamic cytokine profiles of non-vaccinated and primarily-infected subjects reported here provide novel insights into the inflammatory immune landscape in the context of SARS-CoV-2 infection, and offer valuable knowledge for addressing future viral infections and the development of novel treatments