Quantitative analysis of cepharanthine in plasma based on semiautomatic microextraction by packed sorbent combined with liquid chromatography.

The spread of Plasmodium falciparum resistance 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 from Stephania 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 and Plasmodium berghei infected mice. The infection did not impact pharmacokinetic parameters of cepharanthine.

Stability and antiviral activity against human cytomegalovirus of artemisinin derivatives.

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.

Early treatment failure during treatment of Plasmodium falciparum malaria with atovaquone-proguanil in the Republic of Ivory Coast.

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.

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

BACKGROUND: The 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. METHOD: Ninety-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. RESULTS: Significant 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 IC(50) of 682 nM versus median IC(50) of 300 nM; p = 0.0146) while isolates with one NHNDNHNNDDD repeat presented significantly reduced QN susceptibility than those who had two (median IC(50) of 704 nM versus median IC(50) 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 IC(50) showed also the highest percentage of isolates carrying the pfnhe1 haplotype 7. CONCLUSIONS: The 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.

Antimalarial drug use in general populations of tropical Africa.

BACKGROUND: The burden of Plasmodium falciparum malaria has worsened because of the emergence of chloroquine resistance. Antimalarial drug use and drug pressure are critical factors contributing to the selection and spread of resistance. The present study explores the geographical, socio-economic and behavioural factors associated with the use of antimalarial drugs in Africa. METHODS: The presence of chloroquine (CQ), pyrimethamine (PYR) and other antimalarial drugs has been evaluated by immuno-capture and high-performance liquid chromatography in the urine samples of 3,052 children (2-9 y), randomly drawn in 2003 from the general populations at 30 sites in Senegal (10), Burkina-Faso (10) and Cameroon (10). Questionnaires have been administered to the parents of sampled children and to a random sample of households in each site. The presence of CQ in urine was analysed as dependent variable according to individual and site characteristics using a random - effect logistic regression model to take into account the interdependency of observations made within the same site. RESULTS: According to the sites, the prevalence rates of CQ and PYR ranged from 9% to 91% and from 0% to 21%, respectively. In multivariate analysis, the presence of CQ in urine was significantly associated with a history of fever during the three days preceding urine sampling (OR = 1.22, p = 0.043), socio-economic level of the population of the sites (OR = 2.74, p = 0.029), age (2-5 y = reference level; 6-9 y OR = 0.76, p = 0.002), prevalence of anti-circumsporozoite protein (CSP) antibodies (low prevalence: reference level; intermediate level OR = 2.47, p = 0.023), proportion of inhabitants who lived in another site one year before (OR = 2.53, p = 0.003), and duration to reach the nearest tarmacked road (duration less than one hour = reference level, duration equal to or more than one hour OR = 0.49, p = 0.019). CONCLUSION: Antimalarial drug pressure varied considerably from one site to another. It was significantly higher in areas with intermediate malaria transmission level and in the most accessible sites. Thus, P. falciparum strains arriving in cross-road sites or in areas with intermediate malaria transmission are exposed to higher drug pressure, which could favour the selection and the spread of drug resistance.