Véronique Sinou

Synthesis, characterization, and in vitro antimalarial and antitumor activity of new ruthenium(II) complexes of chloroquine.

The new Ru(II) chloroquine complexes [Ru(eta(6)-arene)(CQ)Cl2] (CQ = chloroquine; arene = p-cymene 1, benzene 2), [Ru(eta(6)-p-cymene)(CQ)(H2O)2][BF4]2 (3), [Ru(eta(6)-p-cymene)(CQ)(en)][PF6]2 (en = ethylenediamine) (4), and [Ru(eta(6)-p-cymene)(eta(6)-CQDP)][BF4]2 (5, CQDP = chloroquine diphosphate) have been synthesized and characterized by use of a combination of NMR and FTIR spectroscopy with DFT calculations. Each complex is formed as a single coordination isomer: In 1-4, chloroquine binds to ruthenium in the eta(1)-N mode through the quinoline nitrogen atom, whereas in 5 an unprecedented eta(6) bonding through the carbocyclic ring is observed. 1, 2, 3, and 5 are active against CQ-resistant (Dd2, K1, and W2) and CQ-sensitive (FcB1, PFB, F32, and 3D7) malaria parasites (Plasmodium falciparum); importantly, the potency of these complexes against resistant parasites is consistently higher than that of the standard drug chloroquine diphosphate. 1 and 5 also inhibit the growth of colon cancer cells, independently of the p53 status and of liposarcoma tumor cell lines with the latter showing increased sensitivity, especially to 1 (IC50 8 microM); this is significant because this type of tumor does not respond to currently employed chemotherapies.

New ferrocenic pyrrolo[1,2-a]quinoxaline derivatives: synthesis, and in vitro antimalarial activity.

Following our search for antimalarial compounds, novel series of ferrocenic pyrrolo[1,2-a]quinoxaline derivatives 1-2 were synthesized from various substituted nitroanilines and tested for in vitro activity upon the erythrocytic development of Plasmodiumfalciparum strains with different chloroquine-resistance status. The pyrrolo[1,2-a]quinoxalines 1 were prepared in 6-8 steps through a regioselective palladium-catalyzed monoamination by coupling 4-chloropyrrolo[1,2-a]quinoxalines with 1,3-bis(aminopropyl)piperazine or -methylamine using Xantphos as the ligand. The ferrocenic bispyrrolo[1,2-a]quinoxalines 2 were prepared by reductive amination of previously described bispyrrolo[1,2-a]quinoxalines 9 with ferrocene-carboxaldehyde, by treatment with NaHB(OAc)(3). The best results were observed with ferrocenic pyrrolo[1,2-a]quinoxalines linked by a bis(3-aminopropyl)piperazine. Moreover, it was observed that a methoxy group on the pyrrolo[1,2-a]quinoxaline nucleus and no substitution on the terminal N-ferrocenylmethylamine function enhanced the pharmacological activity. Selected compounds 1b, 1f-h, 1l and 2a were tested for their ability to inhibit beta-haematin formation, the synthetic equivalent of hemozoin, by using the HPIA (heme polymerization inhibitory activity) assay. Of the tested compounds, only 2a showed a beta-haematin formation inhibition, but no inhibition of haem polymerization was observed with the other selected ferrocenic monopyrrolo[1,2-a]quinoxaline derivatives 1b, 1f-h and 1l, as the IC(50) values were superior to 10 equivalents.

Intramuscular Bioavailability and Clinical Efficacy of Artesunate in Gabonese Children with Severe Malaria

ABSTRACT Artesunate (ARS) is a water-soluble artemisinin derivative that is a potential alternative to quinine for the treatment of severe childhood malaria. We studied the pharmacokinetics and bioavailability of ARS given by the intramuscular (i.m.) route in an open crossover study design. Fourteen children were randomized to receive intravenous (i.v.) ARS in a loading dose (2.4 mg/kg of body weight) followed 12 h later by an i.m. dose (1.2 mg/kg) (group I), and 14 children were randomized to receive i.m. ARS (2.4 mg/kg) followed by an i.v. dose of ARS (1.2 mg/kg) (group II). We carried out a two-compartment analysis of ARS and dihydroartemisinin (DHA; the principal antimalarial metabolite) levels in 21 children (groups I and II combined). Absorption of i.m. ARS was rapid, with the maximum concentration of DHA in serum being achieved in less than 1 h in most children (median time to the maximum concentration of drug in serum, 35.1 min; range, 10.8 to 71.9 min). The absolute bioavailability of DHA was a median of 86.4% (range, 11.4 to 462.1%), the median steady-state volume of distribution was 1.3 liters/kg (range, 0.5 to 7.9 liters/kg), and the median clearance was 0.028 liters/kg/min (range, 0.001 to 1.58 liters/kg/min). There were no major adverse events attributable to ARS. Parasite clearance kinetics were comparable between the two treatment groups. These results support the use of i.m. ARS in children with severe malaria.

High-Throughput Analysis of Antimalarial Susceptibility Data by the WorldWide Antimalarial Resistance Network (WWARN) In Vitro Analysis and Reporting Tool

ABSTRACT Assessment of in vitro susceptibility is a fundamental component of antimalarial surveillance studies, but wide variations in the measurement of parasite growth and the calculation of inhibitory constants make comparisons of data from different laboratories difficult. Here we describe a Web-based, high-throughput in vitro analysis and reporting tool (IVART) generating inhibitory constants for large data sets. Fourteen primary data sets examining laboratory-determined susceptibility to artemisinin derivatives and artemisinin combination therapy partner drugs were collated from 11 laboratories. Drug concentrations associated with half-maximal inhibition of growth (IC50s) were determined by a modified sigmoid Emax model-fitting algorithm, allowing standardized analysis of 7,350 concentration-inhibition assays involving 1,592 isolates. Examination of concentration-inhibition data revealed evidence of apparent paradoxical growth at high concentrations of nonartemisinin drugs, supporting amendment of the method for calculating the maximal drug effect in each assay. Criteria for defining more-reliable IC50s based on estimated confidence intervals and growth ratios improved correlation coefficients for the drug pairs mefloquine-quinine and chloroquine-desethylamodiaquine in 9 of 11 and 8 of 8 data sets, respectively. Further analysis showed that maximal drug inhibition was higher for artemisinins than for other drugs, particularly in ELISA (enzyme-linked immunosorbent assay)-based assays, a finding consistent with the earlier onset of action of these drugs in the parasite life cycle. This is the first high-throughput analytical approach to apply consistent constraints and reliability criteria to large, diverse antimalarial susceptibility data sets. The data also illustrate the distinct biological properties of artemisinins and underline the need to apply more sensitive approaches to assessing in vitro susceptibility to these drugs.

Polymorphism of Plasmodium falciparum Na+/H+ exchanger is indicative of a low in vitro quinine susceptibility in isolates from Viet Nam

BackgroundThe Plasmodium falciparum NA+/H+ exchanger (pfnhe1, gene PF13_0019) has recently been proposed to influence quinine (QN) susceptibility. However, its contribution to QN resistance seems to vary geographically depending on the genetic background of the parasites. Here, the role of this gene was investigated in in vitro QN susceptibility of isolates from Viet Nam.MethodNinety-eight isolates were obtained from three different regions of the Binh Phuoc and Dak Nong bordering Cambodia provinces during 2006-2008. Among these, 79 were identified as monoclonal infection and were genotyped at the microsatellite pfnhe1 ms4760 locus and in vitro QN sensitivity data were obtained for 51 isolates. Parasite growth was assessed in the field using the HRP2 immunodetection assay.ResultsSignificant associations were found between polymorphisms at pfnhe1 microsatellite ms4760 and susceptibility to QN. Isolates with two or more DNNND exhibited much lower susceptibility to QN than those harbouring zero or one DNNND repeats (median IC50 of 682 nM versus median IC50 of 300 nM; p = 0.0146) while isolates with one NHNDNHNNDDD repeat presented significantly reduced QN susceptibility than those who had two (median IC50 of 704 nM versus median IC50 of 375 nM; p < 0.01). These QNR associated genotype features were mainly due to the over representation of profile 7 among isolates (76.5%). The majority of parasites had pfcrt76T and wild-type pfmdr1 (> 95%) thus preventing analysis of associations with these mutations. Interestingly, area with the highest median QN IC50 showed also the highest percentage of isolates carrying the pfnhe1 haplotype 7.ConclusionsThe haplotype 7 which is the typical Asian profile is likely well-adapted to high drug pressure in this area and may constitute a good genetic marker to evaluate the dissemination of QNR in this part of the world.

Past and new challenges for malaria control and elimination: the role of operational research for innovation in designing interventions

This meeting report presents the outcomes of a workshop held in Bangkok on December 1st 2014, where the following challenges were discussed: the threat of resistance to artemisinin and artemisinin-based combination therapy in the Greater Mekong Sub-region (GMS) and in Africa; access to treatment for most at risk and hard to reach population; insecticide resistance, residual and outdoors transmission. The role of operational research and the interactions between research institutions, National Malaria Control Programmes, Civil Society Organizations, and of financial and technical partners to address those challenges and to accelerate translation of research into policies and programmes were debated. The threat and the emergency of the artemisinin resistance spread and independent emergence in the GMS was intensely debated as it is now close to the border of India. The need for key messages, based on scientific evidence and information available and disseminated without delay, was highlighted as crucial for an effective and urgent response.

New PfATP6 Mutations Found in Plasmodium falciparum Isolates from Vietnam

Artemisinin and its derivatives have been used against malaria in Vietnam since 1991 (4). An increase in clinical artemisinin resistance would be disastrous for malaria treatment. All possible indicators of this potential resistance must be monitored. The sarco/endoplasmic reticulum Ca2+-ATPase ortholog of Plasmodium falciparum (PfATP6) has been suggested to be the target of artemisinins (3). Consequently, the polymorphism of PfATP6 is being monitored by several scientific research teams (2, 6, 7, 9, 10, 15) We report here the genotyping results of PfATP6 from 98 P. falciparum field isolates collected in 2006 to 2007 in South Vietnam. Parasite samples were taken from patients (28.82 ± 12.31 years old) with uncomplicated P. falciparum infections before drug treatment. They were collected in Binh Phuoc and Dak Nong provinces in South Vietnam. Patients did not follow a chemoprophylaxis before sampling. Diagnosis was carried out by microscopic examination and confirmed by real-time PCR as previously described (14). The whole PfATP6 gene was sequenced once in both directions with five primer pairs (adapted from Jambou et al. [7]) and compared to the reference sequence of the 3D7 strain (“PFA0310c” in the genome annotation). We found a total of eight mutations (Table ​(Table1):1): four nonsynonymous (I89T, N463S, N465S, and N683K), three synonymous (N460N, I898I, and C1031C), and one double deletion leading to the loss of two asparagines (Δ463-464). Five of these have not been described previously (N460N, N463S, Δ463-464, N465S, and C1031C). All of the mutations were detected on different isolates, except for I898I, which was found alone or associated with others. Like Mugittu et al. in Tanzania (10) and Zhang et al. in China (15), we did not find either the S769N mutation or the A623E E431K double mutation, associated with reduced susceptibility to artemether (7). Previously, the N683K mutation was only found in Cambodia (2), suggesting that it may be specific to P. falciparum from South-East Asia. However, we did not detect this mutation in the South-East Asiatic strains W2 and Dd2 (both from Indochina, Malaria Research and Reference Reagent Resource Center), IMT-A4 (Vietnam), and IMT-K2 (Cambodia; data not shown). Interestingly, the N460N, N463S, N465S, and N683K mutations and the Δ463-464 double deletion are in a stretch of nine asparagines located in the interspecies variable region of PfATP6, a domain specific for Plasmodium species (8). Consequently, these modifications could be adaptive changes that might alter susceptibility to artemisinins. TABLE 1. Diversity of PfATP6 in P. falciparum samples from Vietnam Cojean et al. found the S769N mutation in an isolate from Africa that was susceptible to dihydroartemisinin (1), while Noedl et al. did not find this mutation in Cambodian samples that were less susceptible to artesunate (11). Consequently, we speculated on whether the correlation between the S769N mutation and the increased artemether 50% inhibitory concentration found in six isolates from French Guyana (7) should be regarded as a local case. Like other investigators, we did not detect any polymorphism in codon 263, described as the key amino acid for the interaction between PfATP6 and artemisinins (13). Mutations observed in our sequences and in those of previous studies (2, 6, 9) could be implicated indirectly in this interaction, in the case of association with artemisinin susceptibility. Considering the development of artemisinin combined therapies and the possible implication of PfATP6 in artemisinin resistance, the molecular variability of this gene should be carefully monitored.

Diastereoselective synthesis of potent antimalarial cis-β-lactam agents through a [2 + 2] cycloaddition of chiral imines with a chiral ketene

The effect of double asymmetric induction for the synthesis of new cis-β-lactams by [2 + 2] cycloaddition reactions of chiral imines with a chiral ketene was investigated. The cycloaddition reaction was found to be totally diastereoselective leading exclusively to the formation of the cis-β-lactam derivatives. The newly synthesized cycloadducts were evaluated for their antimalarial activities against Plasmodium falciparum K14 resistant strain with moderate to excellent IC50 values varying from 8 to 50 μM. Of the fifteen β-lactams tested, four showed IC50 ≤ 11 μM.

Differential Association of Plasmodium falciparum Na+/H+ Exchanger Polymorphism and Quinine Responses in Field- and Culture-Adapted Isolates of Plasmodium falciparum

ABSTRACT Plasmodium falciparum isolates with decreased susceptibility to quinine are increasingly being found in malaria patients. Mechanisms involved in this resistance are not yet understood. Several studies claim that alongside mutations in the Pfcrt and Pfmdr1 genes, the Pfnhe-1 Na+/H+ exchanger polymorphism plays a role in decreasing susceptibility. However, conflicting results on the link between the Pfnhe-1 gene and quinine resistance arise from field- and culture-adapted isolates. We tested the association between Pfnhe-1, Pfcrt, and Pfmdr1 polymorphisms in field- and culture-adapted isolates from various countries with their in vitro susceptibility to quinine. Field isolates presented a higher diversity of the Pfnhe-1 microsatellite sequence than culture-adapted isolates. In culture-adapted isolates but not in field isolates, mutations in the Pfcrt and Pfmdr1 genes, as well as a higher number of DNNND repeats in the Pfnhe-1 gene, were associated with a higher 50% inhibitory concentration (IC50) of quinine. Furthermore, most of the culture-adapted isolates with more than one DNNND repeat in the Pfnhe-1 gene also harbored mutated Pfcrt and Pfmdr1 genes with an apparent cumulative effect on quinine susceptibility. This study supports the involvement of the Pfnhe-1 gene in the modulation of the in vitro quinine response when associated with mutated Pfcrt and Pfmdr1 genes. Culture adaptation could be responsible for selection of specific haplotypes of these three genes. Methods used for drug testing might thus influence the association between Pfnhe-1 polymorphism and quinine susceptibility. However, we do not exclude the possibility that in particular settings, Pfnhe-1 polymorphism can be used as a molecular marker for surveillance of quinine resistance.

Diversity of Anopheles mosquitoes in Binh Phuoc and Dak Nong Provinces of Vietnam and their relation to disease

BackgroundHuman malaria is still a burden in Dak Nong and Binh Phuoc Provinces in south-central Vietnam that border Cambodia. Several Anopheles species that transmit human malarial Plasmodium may also transmit Wuchereria bancrofti, the nematode that causes Bancroftian lymphatic filariasis. The objective of this study was to investigate the role of Anopheles species in the transmission of these two pathogens in the two highly malaria endemic provinces of Vietnam.MethodsAnopheles mosquitoes were collected in Dak Nong and Binh Phuoc Provinces in November and December of 2010 and 2011. Human landing catches, paired collections on human and buffalo, and resting captures were made with mouth aspirators. Collections were also made with light traps. Morphological and PCR-based methods were used to identify the species. Real-time PCR was used to detect Plasmodium species and W. bancrofti in individual mosquitoes.ResultsTwenty-four Anopheles species were identified among 797 captured mosquitoes. Anopheles dirus was found in both provinces and was the predominant species in Binh Phuoc Province; An. maculatus was the most prevalent species in Dak Nong Province. Anopheles minimus was collected only in Binh Phuoc Province. Some specimens of An. minimus and An. pampanai were misidentified based on morphology. Four specimens of An. scanloni were identified, and this is the first report of this species of the Dirus Complex in Vietnam. Two females, one An. dirus and one An. pampanai, collected in Binh Phuoc Province were infected with P. vivax, for an overall infection rate of 0.41% (2/486): 0.28% for An. dirus (1/361) and 20% for An. pampanai (1/5). No mosquitoes were found to be infected with P. falciparum, P. knowlesi or W. bancrofti in either province.ConclusionA diversity of Anopheles species occurs in Dak Nong and Binh Phuoc Provinces of Vietnam, several of which are considered to be actual and potential vectors of malarial protozoa and microfilariae. It is highly likely that two of the species, An. dirus and An. pampanai, are active in malaria transmission based on the detection of P. vivax in females of these species. This is the first report of An. scanloni in Vietnam.