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Summary L-abrine and ricinine are two small molecules found respectively in the seeds of Abrus precatorius and Ricinus communis. L-abrine and ricinine are extracted simultaneously with the toxins ricin and abrin from the seeds and are therefore exposure markers for these toxins. We developed and validated a fast and simple method for the simultaneous quantitation of ricinine and L-abrine in plasma and urine by ultra-high performance liquid chromatography coupled with a tandem mass spectrometer (UHPLC LC40 Nexera X3-TQ8060 Shimadzu). A pretreatment step was achieved using solid phase extraction on the Strata®-X 30 mg, 1 mL cartridge (Phenomenex) before a chromatographic separation on a Luna Omega Polar C18 column (50 × 2.1 mm, 1.6 μm; Phenomenex) with a gradient of mobile phase composed of water and acetonitrile, each containing 0.1% formic acid. This method is sensitive (lower limit of quantification at 0.4 and 2 ng/mL in plasma and in urine respectively), fast (chromatographic analysis in 5 minutes), easier and cheaper than the proteomic analysis of the toxins. It represents an interesting alternative for highlighting an intoxication by ricin or abrin.

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With millions of intoxications each year and over 200,000 deaths, organophosphorus (OP) compounds are an important public health issue worldwide. OP poisoning induces cholinergic syndrome, with respiratory distress, hypertension, and neuron damage that may lead to epileptic seizures and permanent cognitive deficits. Existing countermeasures are lifesaving but do not prevent long-lasting neuronal comorbidities, emphasizing the urgent need for animal models to better understand OP neurotoxicity and identify novel antidotes. Here, using diisopropylfluorophosphate (DFP), a prototypic and moderately toxic OP, combined with zebrafish larvae, we first showed that DFP poisoning caused major acetylcholinesterase inhibition, resulting in paralysis and CNS neuron hyperactivation, as indicated by increased neuronal calcium transients and overexpression of the immediate early genes fosab, junBa, npas4b, and atf3. In addition to these epileptiform seizure-like events, DFP-exposed larvae showed increased neuronal apoptosis, which were both partially alleviated by diazepam treatment, suggesting a causal link between neuronal hyperexcitation and cell death. Last, DFP poisoning induced an altered balance of glutamatergic/GABAergic synaptic activity with increased NR2B-NMDA receptor accumulation combined with decreased GAD65/67 and gephyrin protein accumulation. The zebrafish DFP model presented here thus provides important novel insights into the pathophysiology of OP intoxication, making it a promising model to identify novel antidotes.

A structure-activity relationship study of active molecules against chloroquine-resistant Plasmodium falciparum K1 strain is reported. Structurally simplified analogues of antiplasmodial active alkaloids presented similar levels of activity as their corresponding natural products extracted from Guiera senegalensis and Mitragyna inermis with IC50 values on chloroquine-resistant P. falciparum K1 strain of up to 10.6 μM for spirooxindoles and 13.8 μM for β-carbolines. The identification of such simpler and cheaper structural analogues is crucial to efficiently study these natural products' action mode as well as developing new cures against malaria.

Sulfur mustard is a chemical warfare agent that damages the eyes, lungs and skin. In the latter, it induces severeburns that classify it as vesicant agents. Highly reactive, sulfur mustard has the ability to alkylate many biomolecules.The products derived from these reactions can be used, mainly using analytical chemistry methods, as biomarkersof exposure or effect. Sulfur mustard also greatly disrupts metabolism and protein induction. These responsescan be used to highlight longer-term effects., L’ypérite est un toxique de guerre endommageant les yeux, les poumons et la peau. Dans cette dernière, elle induitde graves brûlures qui la classent parmi les agents vésicants. Très réactive, elle possède des capacités d’alkylationde nombreuses biomolécules. Les produits qui en dérivent peuvent être utilisés, principalement en employantdes méthodes de chimie analytique, comme biomarqueurs d’exposition ou d’effet. L’ypérite perturbe égalementfortement le métabolisme et l’induction de protéines. Ces réponses sont utilisables pour mettre en évidencedes effets à plus long terme.

Resistance to antibiotics is an emerging phenomenon and a major medical problem. The resistance of Gram-negative bacteria such as Pseudomonas aeruginosa and the Burkholderia group to conventional antibiotics leads to therapeutic failure and requires new antibiotic therapies. The use of iron transport systems is a promising strategy to overcome this phenomenon. These TonB-dependent receptors, essential for the survival of microorganisms, allow specific recognition of ferric siderophore complexes in order to transport iron within bacteria1. Bacteria, according to their kind, express different types of receptors that allow them to recognize their endogenous siderophores but also xenosiderophores. Pseudomonas aeruginosa and Burkholderia pseudomallei in particular possess FptA receptors allowing the recognition of pyochelin.2 These specific systems may allow the introduction of antibacterial agents by forming antibiotic-siderophore conjugates or toxic complexes such as gallium complexes, in the bacteria to kill it. Siderophores have three types of chelating function: catechols, hydroxamates and hydroxy-carboxylates. Previous work in the laboratory has shown that piperazine 1,4-dicatechol structures (MPPS0225) could be recognized by Pseudomonas aeruginosa strains. In order to further investigate this piperazine platform, we have synthesized iron chelators bearing 3-hydroxypyridin-4-ones and 1,3-dihydroxypyridin-4-one ligands. At the same time, we were interested in the synthesis of a more complex 2,5-dioxopiperazine platform, part of the rhodotorulic acid (RA), a natural siderophore produced by Rhodotorula pilimanae showing an interesting iron affinity (pFe = 21,8). Two RA synthesis strategies will be developed as well as the corresponding 3,6-disubstituted analogs. Through the synthesis of these chelators, we would like to study the influence, on the iron complexation, of: i) the nitrogen platform (piperazine or dioxopiperazine), ii) the presence of stereogenic centers (3,6-disubstituted dioxopiperazine vs 1,4 -disubstituted piperazines) and iii) the nature of the iron ligands (hydroxypyridinone vs catechol). An evaluation of the siderophore-like potential and a measurement of the complexing force will be carried out. We would like to thank the DGA and the Haut de France region for their financial support. References Miethke M.; Marahiel MA. Microbiology and Molecular Biology Reviews. 2007, 71, 413-451. Butt AT.; Thomas MS. Frontiers in Cellular and Infection Microbiology. 2017, 7.

Malaria is a neglected tropical disease that remains a leading cause of morbidity and mortality among the world’s poorest populations. In 2015, 91 tropical and sub-tropical countries are endemic for this infectious disease. Pregnant women and children are the most sensitive to this infection and, in 2015, 429 000 people died. Among the five species of Plasmodium responsible for human malaria, P. falciparum is the parasite which causes the most serious form of the disease. More recent efforts focused on the development of antimalarial vaccines and since 2006, World Health Organization (WHO) recommends artemisinin-based combination therapies (ACTs). In drugs resistance areas, several antimalarial drugs, such as aminoaryl-alcohol (mefloquine (MQ), lumefantrine (LM)), are currently used in combination with artemisinin derivatives. However, the emergence of multi-drug-resistant parasites decreases efficacy of ACTs. Thus, the design of new active compounds on Plasmodium-resistant strains is urgently. We have previously developed an asymmetric synthesis to prepare 4-aminoquinoline-methanol enantiomers (AQM) as MQ analogs. They were active on nanomolar range against Pf3D7 (chloroquine-sensitive) and PfW2 (chloroquine-resistant) P. falciparum strains. Interestingly, (S)-enantiomers displayed an activity increased by 2 to 15-fold as compared to their (R)-counterparts. Currently their mechanisms of actions are not totally clear and remain to be explored. In continuation of our work, we are interested now to study the change of heterocycle (fluorene vs quinoline) on the antimalarial activity. We focus on the design and the preparation of novel asymmetric 2,4,7-trisusbtituted fluorenes, new aminofluorene-methanol derivatives (AFM) as LM analogs. The evaluation of their antiplasmodial activities against P. falciparum and their corresponding cytotoxicities proved the interest of this pharmacophore with activities on nanomolar range against Pf3D7 and PfW2. We will present here the access and the biological results on these AFMs.

Organophosphorus nerve agents still represent a serious risk to human health. In the French armed forces, the current emergency treatment against OP intoxications is a fully licensed wet-dry dual-chambered autoinjector (Ineurope ®), that contains pralidoxime methylsulfate (2-PAM) to reactivate inhibited acetylcholinesterase (AChE), atropine sulfate (AS) and avizafone chlorhydrate (AVZ). While this treatment is effective against several of the known nerve agents, it shows little efficacy against the Russian VX (VR), one of the most toxic compounds. HI-6 dimethanesulfonate (HI-6 DMS) is an oxime able to reactivate in vitro and in vivo VR-inhibited AChE. To confirm the superiority of HI-6 DMS towards 2-PAM prior to licensing, we compared the two 3-drug-combinations (HI-6 vs 2-PAM, 33 and 18 mg/kg respectively, equimolar doses; AS/AVZ 0.25/0.175 mg/kg respectively) in VR-poisoned cynomolgus macaques, the model required by the French drug regulatory agency. In parallel we performed HI-6 pharmacokinetics analysis using a one compartment model. A better efficacy of the HI-6 DMS combination was clearly observed: up to 5 LD50 of VR (i.m.), a single administration of the HI-6 DMS combination, shortly after the onset of clinical signs, prevented death of the four intoxicated animals. Conversely 2-PAM only prevented death in one out of three subjects exposed to the same amount of VR. As expected with V agents, reinhibition of blood AChE was observed but without any apparent impact on the clinical recovery of the animals. A single administration of the HI-6 DMS combination was still but partially effective at 15 LD50 of VR, allowing a 50% survival rate.

International audience; Background: We prepared a novel series of enantiopure mefloquine analogues with pyrrolo[1,2-a]quinoxaline core in order to fight Plasmodium falciparum resistant strain. Objectives: To observe the influence of pyrrolo[1,2-a]quinoxaline core versus quinoline core on the antimalarial activity. Method: Four enantiopure aminoalcoholpyrrolo[1,2-a]quinoxalines 2 were synthetized via Sharpless asymmetric dihydroxylation reaction in eight steps. Their antimalarial activity was evaluated on two Plasmodium falciparum strains 3D7 and W2 with a SYBR Green I fluorescence-based method and their cytotoxicity was measured on four cell lines HepG2, THP-1, CHO and HFF. Results: IC50 values of the four compounds 2 were close to the micromolar against the two P. falciparum strains. They were more active against P. falciparum strain W2 vs. P. falciparum strain 3D7. (R)-enantiomers were always more active than their (S)-counterpart whatever the strain. Selectivity indexes of compounds 2 were lower than 100. Conclusion: A novel series of enantiopure aminoalcohols with pyrrolo[1,2-a]quinoxaline core were synthesized in eight steps. They displayed IC50 values close to the micromolar against two P. falciparum strains 3D7 and W2. Although, In this series, 2,8-bistrifluoromethylquinoline was a best core than pyrrolo[1,2-a]quinoxaline for an optimal antimalarial activity, the pyrroloquinoxaline 2b showed an interesting antimalarial activity.

Albitiazolium is the lead compound of bisthiazolium choline analogues and exerts powerful in vitro and in vivo antimalarial activities. Here we provide new insight into the fate of albitiazolium in vivo in mice and how it exerts its pharmacological activity. We show that the drug exhibits rapid and potent activity and has very favorable pharmacokinetic and pharmacodynamic properties. Pharmacokinetic studies in Plasmodium vinckei-infected mice indicated that albitiazolium rapidly and specifically accumulates to a great extent (cellular accumulation ratio, >150) in infected erythrocytes. Unexpectedly, plasma concentrations and the area under concentration-time curves increased by 15% and 69% when mice were infected at 0.9% and 8.9% parasitemia, respectively. Albitiazolium that had accumulated in infected erythrocytes and in the spleen was released into the plasma, where it was then available for another round of pharmacological activity. This recycling of the accumulated drug, after the rupture of the infected erythrocytes, likely extends its pharmacological effect. We also established a new viability assay in the P. vinckei-infected mouse model to discriminate between fast- and slow-acting antimalarials. We found that albitiazolium impaired parasite viability in less than 6 and 3 h at the ring and late stages, respectively, while parasite morphology was affected more belatedly. This highlights that viability and morphology are two parameters that can be differentially affected by a drug treatment, an element that should be taken into account when screening new antimalarial drugs.

Ethnopharmacological relevance Stephania rotunda Lour. (Menispermaceae) is an important traditional medicinal plant that is grown in Southeast Asia. The stems, leaves, and tubers have been used in the Cambodian, Lao, Indian and Vietnamese folk medicine systems for years to treat a wide range of ailments, including asthma, headache, fever, and diarrhoea. Aim of the review: To provide an up-to-date, comprehensive overview and analysis of the ethnobotany, phytochemistry, and pharmacology of Stephania rotunda for its potential benefits in human health, as well as to assess the scientific evidence of traditional use and provide a basis for future research directions. Material and methods Peer-reviewed articles on Stephania rotunda were acquired via an electronic search of the major scientific databases (Pubmed, Google Scholar, and ScienceDirect). Data were collected from scientific journals, theses, and books. Results The traditional uses of Stephania rotunda were recorded in countries throughout Southeast Asia (Cambodia, Vietnam, Laos, and India). Different parts of Stephania rotunda were used in traditional medicine to treat about twenty health disorders. Phytochemical analyses identified forty alkaloids. The roots primarily contain l-tetrahydropalmatine (l-THP), whereas the tubers contain cepharanthine and xylopinine. Furthermore, the chemical composition differs from one region to another and according to the harvest period. The alkaloids exhibited approximately ten different pharmacological activities. The main pharmacological activities of Stephania rotunda alkaloids are antiplasmodial, anticancer, and immunomodulatory effects. Sinomenine, cepharanthine, and l-stepholidine are the most promising components and have been tested in humans. The pharmacokinetic parameters have been studied for seven compounds, including the three most promising compounds. The toxicity has been evaluated for liriodenine, roemerine, cycleanine, l-tetrahydropalmatine, and oxostephanine. Conclusion Stephania rotunda is traditionally used for the treatment of a wide range of ailments. Pharmacological investigations have validated different uses of Stephania rotunda in folk medicine. The present review highlights the three most promising compounds of Stephania rotunda , which could constitute potential leads in various medicinal fields, including malaria and cancer.

The tuber of Stephania rotunda is used to treat malaria in traditional medicine in many Asian countries. Cepharanthine is a bioactive bisbenzylisoquinoline alkaloid of the plant, in particular it presented antiplasmodial activity. Two modern and ecologic technologies were proposed to extract cepharanthine from S. rotunda tuber: ultrasound-assisted extraction (UAE) and microwave-assisted extraction (MAE). Various parameters of these processes were studied to optimize the extraction of cepharanthine from the tuber. Quantification of cepharanthine was performed by a validated HPLC method. MAE in closed system, for 15 min, with the ratio solid–liquid 1/30, the solvent ethanol–water (50:50, v/v), at 80 °C and 100 W, was the most effective conditions on the extraction yield. UAE was the most ecologic because it did not need any heating and was shorter than MAE (10 min). Statistical analyses showed that these two methods exhibited the same efficiency for the extraction of cepharanthine and both methods were more efficient than percolation. MAE and UAE permitted the best extraction of cepharanthine from the S. rotunda tuber when the solvent used was ethanol–water in comparison with dichloromethane. Both eco-extractions, using ethanol–water, were found to be promising alternative methods for the extraction of cepharanthine from S. rotunda.

Objectives Artesunate, a derivative of dihydroartemisinin, itself a product of artemisinin, inhibits the replication of cytomegalovirus in vitro. In vivo, artesunate undergoes rapid conversion into the active metabolite dihydroartemisinin. The in vitro stability of the compounds and the antiviral activity of dihydroartemisinin are of great concern for the interpretation of in vitro testing. The aim of the study was to measure artesunate conversion into dihydroartemisinin in culture medium and to evaluate the stability and antiviral activity of artemisinin derivatives, according to culture conditions. Methods Conversion of artesunate into dihydroartemisinin was measured in culture medium with or without fetal calf serum, in the presence or absence of fibroblast monolayers, at different times. The stability of artemisinin derivatives was determined in serum-enriched medium. Concentrations of each compound inhibiting viral DNA synthesis by 50% were determined in fibroblasts cultured in serum-free or serum-enriched medium, after addition of compound as a single dose or fractional doses. Results Conversion of artesunate into dihydroartemisinin in serum-free or serum-enriched medium was non-equimolar. The half-lives of artesunate, dihydroartemisinin and artemisinin were 10.3 ± 0.9, 5.2 ± 0.5 and 11.2 ± 1.2 h, respectively. Activity of dihydroartemisinin and artesunate was markedly reduced in serum-starved cells. Unexpectedly, dihydroartemisinin displayed a lower activity than artesunate. Addition of both compounds as fractional doses increased their activity. Artemisinin had no anticytomegaloviral activity. Conclusions Artemisinin derivatives were shown to be unstable in vitro and their addition as fractional doses could partly compensate for this instability. Importantly, the cellular physiological condition was a determinant of their antiviral activity.

Falciparum malaria is a potentially deadly infectious disease, imposing a sure and fast biologic diagnosis, an early and efficient treatment. We report a case of severe imported Falciparium malaria who received artesunate, and we rewiew the different diagnostic methods of malaria as well as the clinico-biological characteristics of severe malaria. Recent data concerning malaria treatment are presented, as a pharmacokinetic study leaded during this case.

A new fixed-dose combination of artesunate (AS) plus amodiaquine (AQ) (TRIMALACT) was recently developed for the treatment of uncomplicated falciparum malaria. The originality of this combination lies in its galenic formulation which consists of a three-layer tablet with two layers containing each of the active ingredients, i.e. AS and AQ, and these are separated by a middle layer containing an antioxidant compound. To evaluate the efficacy and tolerability of this combination, adults with uncomplicated malaria received three administrations of two tablets (100:300 mg AS/AQ) in a 24-h interval, in Democratic Republic of Congo. Parasitemia and fever were measured and the plasma levels of parent compounds and metabolites [dihydroartemisinin (DHA) and monodesethylamodiaquine (MdAQ)] were determined by high-performance liquid chromatography. In addition, we determined the prevalence of molecular markers of resistance to chloroquine (CQ) and sulfadoxine/pyrimethamine (SP). The AS/AQ combination TRIMALACT demonstrated a good efficacy resulting in an excellent clinical and parasitological response rate of 100% after correction for PCR results. Treatment regimen was well tolerated. The main disposition parameters to AS+AQ were: for DHA, AUC = 632 +/- 475 ng h/ml and Cmax = 432 +/- 325 ng/ml, and for MdAQ = 14268 +/- 4114 ng h/ml and Cmax = 336 +/- 225 ng/ml (mean +/- standard deviation). Parasite genotyping show high frequencies of molecular SP- and CQ-resistance markers with more 80% of the samples showing more than three mutations linked to SP resistance and 93.48% carrying parasite with the CQ-resistant haplotype. This study shows that the AS/AQ combination TRIMALACT is safe and effective in the treatment of highly drug-resistant falciparum malaria.

Four series of carbazole derivatives, including N-substituted-hydroxycarbazoles, oxazinocarbazoles, isoxazolocarbazolequinones, and pyridocarbazolequinones, were studied using diverse biological test methods such as a CE-based assay for CK2 activity measurement, a cytotoxicity assay with IPC-81 cell line, determination of MIC of carbazole derivatives as antibacterial agents, a Plasmodium falciparum susceptibility assay, and an ABCG2-mediated mitoxantrone assay. Two oxazinocarbazoles Ib and Ig showed CK2 inhibition with IC50 = 8.7 and 14.0 µM, respectively. Further chemical syntheses were realized and the 7-isopropyl oxazinocarbazole derivative 2 displayed a stronger activity against CK2 (IC50 = 1.40 µM). Oxazinocarbazoles Ib, Ig, and 2 were then tested against IPC-81 leukemia cells and showed the ability to induce leukemia cell death with IC50 values between 57 and 62 μM. Further investigations were also reported on antibacterial and antiplasmodial activities. No significant inhibitory activity on ABCG2 efflux pump was detected.

Liquid chromatography-electrospray ionization mass spectrometry methods are described for the simultaneous quantification of a bis-thiazolium compound (T3), its related prodrug (TE3) and an intermediate compound (mTE3) that appeared during the prodrug/drug conversion process, in human plasma, whole blood and red blood cells (RBCs). The methods involve solid phase extraction (SPE) of the compounds and the internal standard (verapamil) from the three different matrices using OasisHLB columns with an elution solvent of 2x1 ml of acetonitrile containing 1 ml/l trifluoroacetic acid (TFA). HPLC separation was performed on a C18 encapped Xterra column packed with 3.5 microm particles. The mobile phase used a 8 min gradient, from water containing 1 ml/l TFA to acetonitrile containing 1 ml/l TFA, at a flow rate of 400 microl/min. Verapamil and the TE3 compound were characterized by the protonated molecules at m/z 455 and m/z 541, respectively. The mTE3 species was detected through the (M)+ ion at m/z 497. The T3 compound was detected by use of two ions, the quaternary ammonium salt (M2+/2) at m/z 227.3 and by the adduct with TFA (M+TFA)+ at m/z 567.3. The drug/internal standard peak area ratios were linked via a quadratic relationship to plasma (or whole blood) concentrations in the tested range of 6.4-1282 microg/l (12.8-2564 microg/kg) for T3, 20-2000 microg/l (40-4000 microg/kg) for mTE3 and 10-2000 microg/l (40-4000 microg/kg) for TE3, and to T3 concentrations in RBCs ranging from 12.8 to 2564 microg/kg. Inter-assay precision (in terms of R.S.D.) was below 13.5% and accuracy ranged from 95.4 to 107%. The dilution of the samples (plasma or whole blood) has no influence on the performance of the methods. The extraction recoveries averaged 87% for T3, 53% for mTE3 and 79% for TE3 in plasma; 79% for T3, 57% for mTE3 and 65% for TE3 in blood; and 93% for T3 in RBCs, and was constant across the calibration range. The lower limits of quantitation were 6.4 microg/l for T3, 20 microg/l for mTE3 and 10 microg/l for TE3 in plasma; 12.8 microg/kg for T3 and 40 microg/kg for mTE3 and TE3 in blood; and 12.8 microg/kg for T3 in RBCs. Stability tests under various conditions were also investigated. The three-step SPE procedure (loading, clean-up, and elution) described in this paper to quantify these new anti-malarial compounds in plasma, whole blood and RBCs, can easily be automated by using either robotisation or an automated sample preparation system.

To the Editor: Resistance of Plasmodium falciparum to antimalarial drugs is one of the most worrying problems in tropical medicine. For P. falciparum malaria acquired in French Guiana, the combination of quinine and doxycycline is one of the first-line recommended treatments (1). Since 1996, only 2 treatment failures with quinine have been reported from that country (2). An elevated 50% inhibitory concentration (IC50), classified as in vitro quinine resistance, was reported for 17% of 32 P. falciparum isolates obtained during 1983–1987 in French Guiana (3). Throughout 1994–2005, isolates were susceptible to quinine, with a mean IC50

International audience; Background: As resistance to marketed anti-malarial drugs continues to spread, the need for new molecules active on Plasmodium falciparum-resistant strains grows. Pure (S) enantiomers of amino-alcohol quinolines previously displayed a good in vitro anti-malarial activity. Therefore, a more thorough assessment of their potential clinical use through a rodent model and an in vitro evaluation of their combination with artemisinin was undertaken. Methods: Screening on a panel of P. falciparum clones with varying resistance profiles and regional origins was performed for the (S)-pentyl and (S)-heptyl substituted quinoline derivatives, followed by an in vitro assessment of their combination with dihydroartemisinin (DHA) on the 3D7 clone and an in vivo assay in a mouse model infected with Plasmodium berghei. Their haemolytic activity was also determined. Results: A steady anti-malarial activity of the compounds tested was found, whatever the resistance profile or the regional origin of the strain. (S)-quinoline derivatives were at least three times more potent than mefloquine (MQ), their structurally close parent. The in vitro combination with DHA yielded an additive or synergic effect for both that was as good as that of the DHA/MQ combination. In vivo, survival rates were similar to those of MQ for the two compounds at a lower dose, despite a lack of clearance of the parasite blood stages. A 50% haemolysis was observed for concentrations at least 1,000-fold higher than the antiplasmodial IC 50 s. Conclusions: The results obtained make those two (S)-amino-alcohol quinoline derivatives good candidates for the development of new artemisinin-based combination therapy (ACT), hopefully with fewer neurologic side effects than those currently marketed ACT, including MQ.

Background New classes of anti-malarial drugs are needed to control the alarming Plasmodium falciparum resistance toward current anti-malarial therapy. The ethnopharmacological approach allows the discovery of original chemical structures from the vegetable biodiversity. Previous studies led to the selection of a bisbenzylisoquinoline, called cepharanthine and isolated from a Cambodian plant: Stephania rotunda. Cepharanthine could exert a mechanism of action different from commonly used drugs. Potential plasmodial targets are reported here. Methods To study the mechanism of action of cepharanthine, a combined approach using phenotypic and transcriptomic techniques was undertaken. Results Cepharanthine blocked P. falciparum development in ring stage. On a culture of synchronized ring stage, the comparisons of expression profiles showed that the samples treated with 5 μM of cepharanthine (IC90) were significantly closer to the initial controls than to the final ones. After a two-way ANOVA (p-value

Background: Stephania rotunda is used by traditional health practitioners in Southeast Asia to treat a wide range of diseases and particularly symptoms related to malaria. Cepharanthine (CEP) is an alkaloid isolated from this plant with potential innovative antiplasmodial activity. The analysis of interactions between antiplasmodial drugs is necessary to develop new drugs combinations to prevent de novo emergence of resistance. The objective of this study was to evaluate the anti-malarial activity of CEP in combination with usual anti-malarial compounds, both in vitro and in vivo. Methods: A fixed ratio method using the isotopic micro test was performed on the chloroquine-resistant plasmodial strain W2 to build isobolograms from eight CEP-based combinations with standard anti-malarial drugs. The efficacy of two combinations was then evaluated in the BALB/c mouse infected with Plasmodium berghei ANKA strain. Results: In vitro, efficiency gains were observed when CEP was combined with chloroquine (CQ), lumefantrine (LUM), atovaquone (ATO), piperaquine (PPQ) and particularly monodesethylamodiaquine (MdAQ), whereas an antagonistic interaction was observed with dihydroartemisinin (DHA) and mefloquine (MQ). In vivo ,t he combination of CEP with CQ or amodiaquine (AQ) improved significantly the survival of mice and extended the delay for parasitic recrudescence. Conclusion: All these observations suggest that CEP could be an interesting lead compound in the development of a combination therapy against malaria.

The spread ofPlasmodium falciparumresistance toward most of the used drugs requires new antimalarial compounds. Taking advantage of the biodiversity, the ethnopharmacological approach opens the way for the discovery and the characterization of potent original molecules. Previous works led to the selection of a bisbenzylisoquinoline, cepharanthine, extracted fromStephania rotunda, which is mainly present in Cambodia. A sensitive and selective liquid chromatography method has been developed for the determination of cepharanthine in mouse plasma. The method involved a semiautomated microextraction by packed sorbent (MEPS) using 4 mg of solid phase silica-C8 sorbent. LC separation was performed on a Kinetex XB-C18 column (2.6 µm) with a mobile phase of acetonitrile containing formic acid and 10 mM ammonium formate buffer pH 3.5. Data were acquired at 282 nm with a diode array detector. The drug/internal standard peak area ratios were linked via linear relationships to plasma concentrations (75–2,000 ng/mL). Precision was below 5% and accuracy was 99.0–102%. Extraction recovery of cepharanthine was 56–58%. The method was successfully used to determine the pharmacokinetic profile of cepharanthine in healthy andPlasmodium bergheiinfected mice. The infection did not impact pharmacokinetic parameters of cepharanthine.

To the Editor: Doxycycline is an effective antimalarial prophylactic drug when administered as a monotherapy 1 day before, daily during, and for 4 weeks after travel to an area where malaria is endemic (1). Doxycycline is currently a recommended chemoprophylactic regimen for travelers visiting areas where malaria is endemic and has a high prevalence of chloroquine or multidrug resistance (2). The World Health Organization also recommends doxycycline in combination with quinine or artesunate as the second-line treatment for uncomplicated Plasmodium falciparum malaria (3). Prophylactic and clinical failures of doxycycline against P. falciparum have been associated with both inadequate doses (4) and poor patient compliance (5). However, resistance can also explain failures of prophylaxis. Cycline resistance in Plasmodium spp. has been documented as a consequence of selective drug pressure in a P. berghei murine malaria model (6). The administration of increasing doses of minocycline to mice infected with 1 × 107 parasites for 86 successive passages over 600 days made it possible to obtain a resistant P. berghei strain with a median drug inhibitory concentration (IC50) of 600 mg/kg/d, which is 6-fold higher than that of the susceptible starting strain (100 mg/kg/d) (6). A Bayesian mixture modeling approach identified 3 different phenotypes (low, medium, and high doxycycline IC50 phenotypic groups) among P. falciparum clinical isolates (7,8). Using 90 isolates from 14 countries, we demonstrated that increases in copy numbers of P. falciparum metabolite drug transporter gene (Pfmdt, PFE0825w) and P. falciparum GTPase TetQ gene (PfTetQ, PFL1710c) are associated with reduced susceptibility to doxycycline (9); this association was later confirmed (7). In addition, isolates with PfTetQ KYNNNN motif repeats are associated with in vitro reduced susceptibility to doxycycline and with a significantly higher probability of having an IC50 above the doxycycline resistance threshold of 35 μM (9,10). We report a case of documented malaria prophylactic failure with doxycycline in a 26-year-old soldier from France who was infected during a 6-week peacekeeping mission in the Central African Republic in 2014. According to his colleagues and the collective prophylaxis intake, the patient had been compliant with doxycycline prophylaxis. On admission to a hospital in Bangui, Central African Republic, the patient had fever (temperature 40°C), alteration of consciousness, and hypotension. The diagnosis of severe P. falciparum malaria was made on the basis of a rapid diagnostic test confirmed by a blood smear test (parasitemia 8% on day 0). Intravenous artesunate was immediately started, in accordance with World Health Organization recommendations (3). The patient’s clinical condition worsened, and kidney failure developed. Twenty-four hours later (day 1), he was transported by airplane to Begin Military Teaching Hospital (Saint-Mande, France). On admission, he had cerebral edema and a P. falciparum parasitemia level of 0.7%. The patient died 1 day later (day 2). A blood sample obtained from the patient on day 1 in France showed a doxycycline concentration of 195 μg/mL plasma. This concentration, which was determined by liquid chromatography coupled with tandem mass spectrometry, was compatible with a last doxycycline uptake 1 day before diagnosis (day −1). The finding of the expected doxycycline plasma concentration, together with assurances (colleague’s statements and collective intake of doxycycline) that the patient had followed the drug regimen, was sufficient to suggest prophylaxis failure in a treatment-compliant patient. The P. falciparum sample obtained from the patient on arrival in France was evaluated for in vitro susceptibility to doxycycline, but the evaluation was unsuccessful. The number of copies of PfTetQ and Pfmdt genes were evaluated relative to the single-copy P. falciparum β-tubulin gene (pfβtubulin), as previously described (7,8). The sample was assayed in triplicate. The 2–ΔΔCt method (where Ct indicates cycle threshold) of relative quantification was used and adapted to estimate the number of copies of Pfmdt and PfTetQ by using the formula ΔΔCt = (Ct (PfTetQ or Pfmdt) − Ct (Pfβtubulin))Sample – (Ct (PfTetQ or Pfmdt) − Ct (Pfβtubulin))Calibrator. Genomic DNA extracted from 3D7 P. falciparum, which has a single copy of each gene, was used for calibrator sample; Pfβtubulin served as the control housekeeping gene. The experiment was assayed twice. The sample had 2 copies of PfTetQ and Pfmdt genes, which suggested decreased in vitro susceptibility of the sample to doxycycline (8,9). The genotyping of PfTetQ sequence polymorphisms was done by using conventional methods with the primers PfTetQ forward (5′-TCACGACAAATGTGCTAGATAC-3′) and PfTetQ reverse (5′-ATCATCATTTGTGGTGGATAT-3′), as previously described (10). Two PfTetQ KYNNNN motif repeats were found in the sample; 35 μM (odds ratio 15) (10). The 2 copies of Pfmdt and the 2 KYNNNN motif repeats have been shown to be associated with parasites with in vitro resistance to doxycycline (9,10). The association of doxycycline resistance (prophylactic failure with statement of correct intake and the presence of an expected concentration) with increased Pfmdt copies and decreased PfTetQ KYNNNN motif repeats suggest that these molecular markers are predictive markers of doxycycline resistance that can be used for resistance surveillance.

Background Dihydroartemisinin-piperaquine is a new ACT that is administered as single daily dose for three days and has been demonstrated to be tolerated and highly effective for the treatment of uncomplicated Plasmodium falciparum malaria. Piperaquine was used alone to replace chloroquine as the first-line treatment for uncomplicated malaria in China in response to increasing chloroquine resistance in the 1970s. However, the rapid emergence of piperaquine-resistant strains that resulted in the cessation of its use in China in the 1980s, suggests that there is cross-resistance between piperaquine and chloroquine. Very few data are available on cross-resistance between piperaquine and chloroquine, and the data that do exist are often contradictory. Methods In total, 280 P. falciparum isolates, collected between April 2008 and June 2012 from patients hospitalized in France with imported malaria from a malaria-endemic country, were assessed ex vivo for piperaquine and chloroquine susceptibilities by using the standard 42-hour 3H-hypoxanthine uptake inhibition method. The chloroquine resistance-associated mutation K76T in pfcrt was also investigated for the 280 isolates. Results The IC50 for piperaquine ranged from 9.8 nM to 217.3 nM (mean = 81.3 nM. The IC50 for chloroquine ranged from 5.0 nM to 1,918 nM (mean = 83.6 nM. A significant but low correlation was observed between the Log IC50 values for piperaquine and chloroquine (r = 0.145, p Pfcrt K76 wild-type group (no = 125) and 87.7 nM in the 76 T mutant group (no = 155). This difference was not significant (p = 0.875, Mann Whitney U test). Conclusions The present work demonstrates that there was no cross-resistance between piperaquine and chloroquine among 280 P. falciparum isolates and that piperaquine susceptibility is not associated with pfcrt, the gene involved in chloroquine resistance. These results confirm the efficacy of piperaquine in association with dihydroartemisinin and support its use in areas in which parasites are resistant to chloroquine.

The increased spread of drug-resistant malaria highlights the need for alternative drugs for treatment and chemoprophylaxis. The combination of atovaquone‐proguanil (Malarone®) has shown high efficacy against Plasmodium falciparum with only mild side-effects. Treatment failures have been attributed to suboptimal dosages or to parasite resistance resulting from a point mutation in the cytochrome b gene. In this paper, a case of early treatment failure was reported in a patient treated with atovaquone-proguanil; this failure was not associated with a mutation in the parasite cytochrome b gene, with impaired drug bioavailability, or with re-infection.

In Africa, Mitragyna inermis (Willd.) O. Kuntze (Rubiaceae) is commonly used in traditional medicine to treat malaria. Antimalarial activity is mostly due to the hydromethanolic extract of M. inermis leaves and especially to the main alkaloids, uncarine D and isorhynchophilline. In the present study, we describe for the first time an HPLC method for the simultaneous quantification of uncarine D and isorhynchophylline in biological matrices. SPE was used to extract the components and the internal standard naphthalene from human and pig plasma samples. Chromatographic separation was performed on a C-18 reversed column at a flow rate of 1 mL/min, using methanol-phosphate buffer (10:90, pH 7), as a mobile phase. Good linearity was observed over the concentration ranges of 0.0662-3.31 microg/mL for uncarine D and 0.0476-2.38 microg/mL for isorynchophylline. The precision was less than 12% and the accuracy was from 86 to 107% without any discrepancy between the two species. Uncarine D and isorhynchophylline recoveries were over 80%. These results allowed the quantification of both uncarine D and isorhynchophylline in pig plasma after intravenous administration of M. inermis extract.

International audience; A new class of antimalarial drugs targeting phospholipid metabolism of the malarial parasite is now in development. In the strategy of this development, two mono-thiazolium salts, T1 and T2, need to be monitored. A liquid chromatography-mass spectrometry (LC-MS) method has been developed and validated according to FDA guidelines for simultaneous determination of T1 and T2 in plasma, whole blood and red blood cells (RBCs) from human and rat. The sample-pre-treatment procedure involved solid phase extraction after protein precipitation. Chromatography was carried out on a Zorbax eclipse XDB C8 column and mass spectrometric analysis was performed using an Agilent 1,100 quadrupole mass spectrometer working with an electrospray ionization source. LC-MS data were acquired in single ion monitoring mode at m/z 312, 326 and 227 for T1, T2 and the internal standard (T3), respectively. The drug/internal standard peak area ratios were linked via a quadratic relationship to concentrations (human and rat plasma: 2.25-900 microg/l; human blood and rat RBCs: 4.5-900 microg/kg). Precision was below 14.5% for T1 and below 13% for T2. Accuracy was 92.6-111% for T1 and 95.6-108% for T2. Extraction recoveries were >or=85% in plasma and >or=53% in blood and RBCs. For T1 and T2, the lower limits of quantitation were 2.25 microg/l in plasma, and 4.5 microg/kg in whole blood and RBCs. Stability tests under various conditions were also investigated. This highly specific and sensitive method was useful to analyse samples from pharmacokinetic studies carried out in rat and would also be useful in clinical trials at a later stage.

OBJECTIVES The aim was to study the pharmacokinetic profile of artesunate and its metabolite dihydroartemisinin (DHA) in a pig model. METHODS Thirteen pigs received either intravenous (iv) or intramuscular (im) artesunate (60 mg), with the alternative preparation given 24 h later in an open crossover design. Five of them also received an additional intra-arterial (ia) artesunate dose (60 mg). The plasma concentrations of artesunate and DHA were determined by high-performance liquid chromatography with electrochemical detection. Population modelling was performed with NONMEM, using a two-compartment model. RESULTS Plasma concentration-time profiles were comparable to those observed in humans, with a rapid and biphasic decline for both artesunate and DHA. Following an iv bolus, artesunate had a median maximum plasma concentration (C(max)) of 13.8 microM [interquartile range (IQR), 10.4-22.1 microM], elimination half-life (t(1/2)) = 18 min (IQR, 16-22 min), total plasma clearance (CL) = 5.58 L/h/kg (IQR, 3.31-5.91 L/h/kg) and volume of distribution (V(d)) = 1.85 L/kg (IQR, 1.27-3.20 L/kg). The median C(max) value for DHA was 3.30 microM (IQR, 2.08-5.95 microM), t(1/2) = 26 min (IQR, 23-31 min), CL/Fm = 4.37 L/h/kg (IQR, 3.29-6.87 L/h/kg) and V(d)/Fm = 2.56 L/kg (IQR, 1.93-4.49 L/kg). Artesunate and DHA pharmacokinetic parameters were similar after ia administration. Following im dosing, median artesunate C(max) was 4.81 microM (IQR, 3.74-5.40 microM), t(1/2) = 18 min (IQR, 16-28 min), CL = 4.37 L/h/kg (IQR, 4.13-4.68 L/h/kg) and V(d) = 2.07 L/kg (IQR, 1.83-2.79 L/kg); the bioavailability was 100%. For DHA, median C(max) was 1.43 microM (IQR, 1.00-1.92 microM), t(1/2) = 27 min (IQR, 25-37 min), CL/Fm = 4.68 L/h/kg (IQR, 3.35-6.73 L/h/kg) and V(d)/Fm = 3.31 L/kg (IQR, 2.89-4.27 L/kg). CONCLUSIONS The pharmacokinetic properties of artesunate and DHA in pigs were similar to those reported in humans, suggesting that the swine model is suitable for determining the preclinical pharmacokinetics of artemisinin derivatives.

A new approach to malarial chemotherapy based on quaternary ammonium that targets membrane biogenesis during intraerythrocytic Plasmodium falciparum development has recently been developed. To increase the bioavailability, nonionic chemically modified prodrugs were synthesized. In this paper, the pharmacological properties of a bisthiazolium salt (T3) and its bioprecursor (TE3) were studied. Their antimalarial activities were determined in vitro against the growth of P. falciparum and in vivo against the growth of P. vinckei in mice. Pharmacokinetic evaluations were performed after T3 (1.3 and 3 mg/kg of body weight administered intravenously; 6.4 mg/kg administered intraperitoneally) and TE3 (1.5 and 3 mg/kg administered intravenously; 12 mg/kg administered orally) administrations to rats. After intraperitoneal administration, very low doses offer protection in a murine model of malaria (50% efficient dose [ED 50 ] of 0.2 to 0.25 mg/kg). After oral administration, the ED 50 values were 13 and 5 mg/kg for T3 and TE3, respectively. Both compounds exerted antimalarial activity in the low nanomolar range. After TE3 administration, rapid prodrug-drug conversion occurred; the mean values of the pharmacokinetic parameters for T3 were as follows: total clearance, 1 liter/h/kg; steady-state volume of distribution, 14.8 liters/kg; and elimination half-life, 12 h. After intravenous administration, T3 plasma concentrations increased in proportion to the dose. The absolute bioavailability was 72% after intraperitoneal administration (T3); it was 15% after oral administration (TE3). T3 plasma concentrations (8 nM) 24 h following oral administration of TE3 were higher than the 50% inhibitory concentrations for the most chloroquine-resistant strains of P. falciparum (6.3 nM).

Background A better anti-malarial efficiency and lower neurotoxicity have been reported for mefloquine (MQ) (+)- enantiomer. However, the importance of stereoselectivity remains poorly understood as the anti-malarial activity of pure enantiomer MQ analogues has never been described. Building on these observations, a series of enantiopure 4-aminoalcohol quinoline derivatives has previously been synthesized to optimize the efficiency and reduce possible adverse effects. Their in vitro activity on Plasmodium falciparum W2 and 3D7 strains is reported here along with their inhibition of β-haematin formation and peroxidative degradation of haemin, two possible mechanisms of action of anti-malarial drugs. Results The (S)-enantiomers of this series of 4-aminoalcohol quinoline derivatives were found to be at least as effective as both chloroquine (CQ) and MQ. The derivative with a 5-carbon side-chain length was the more efficient on both P. falciparum strains. (R )-enantiomers displayed an activity decreased by 2 to 15-fold as compared to their (S) counterparts. The inhibition of β-haematin formation was significantly stronger with all tested compounds than with MQ, irrespective of the stereochemistry. Similarly, the inhibition of haemin peroxidation was significantly higher for both (S) and (R)-enantiomers of derivatives with a side-chain length of five or six carbons than for MQ and CQ. Conclusions The prominence of stereochemistry in the anti-malarial activity of 4-aminoalcohol quinoline derivatives is confirmed. The inhibition of β-haematin formation and haemin peroxidation can be put forward as presumed mechanisms of action but do not account for the stereoselectivity of action witnessed in vitro.

Introduction Malaria is a notifiable disease in all European Union and European Economic Area countries except Belgium and France, where only autochthonous malaria is notifiable. Although morbidity caused by malaria has been assessed, little is known about mortality incidence. Objective Our aim was to estimate the number of imported malaria-related deaths in hospital in metropolitan France. Methods We matched individual deaths reported between 1 January 2005 and 31 December 2014 to the French National Reference Centre for malaria (FNRCm) with malaria-related deaths from two other sources: the French National Registry on medical causes of death and the French national hospital discharge database. A capture–recapture method with log-linear modelling was used. Age, sex and place of death stratification were applied to remove heterogeneity. Results The estimated malaria-related deaths in metropolitan France during the study period were 205 (95% confidence interval (CI): 191–219). The annual mean number of malaria-related deaths was estimated at 21 (95% CI: 19–22). The FNRCm malaria-related deaths surveillance had a 38% sensitivity (95% CI: 32–44). Among 161 in-hospital individual malaria-related deaths reported from three data sources, the sex ratio (male to female) was 2.6. Median age of the patients was 57 years, ranging from 1 to 89 years. Conclusion The pertinent finding of this report is that malaria-related death records were significantly less* complete than case records. Therefore, data comparison of imported malaria morbidity and mortality between countries should imperatively be assessed using standard indicators weighted according to the completeness of health surveillance systems.