Highly active ozonides selected against drug resistant malaria

Ever increasing multi-drug resistance by Plasmodium falciparum is creating new challenges in malaria chemotherapy. In the absence of licensed vaccines, treatment and prevention of malaria is heavily dependent on drugs. Potency, range of activity, safety, low cost and ease of administration are crucial issues in the design and formulation of antimalarials. We have tested three synthetic ozonides NAC89, LC50 and LCD67 in vitro and in vivo against multidrug resistant Plasmodium. In vitro, LC50 was at least 10 times more efficient inhibiting P. falciparum multidrug resistant Dd2 strain than chloroquine and mefloquine and as efficient as artemisinin (ART), artesunate and dihydroartemisinin. All three ozonides showed high efficacy in clearing parasitaemia in mice, caused by multi-drug resistant Plasmodium chabaudi strains, by subcutaneous administration, demonstrating high efficacy in vivo against ART and artesunate resistant parasites.

Partial characterization of Plasmodium falciparum trophozoite proteome under treatment with quinine, mefloquine and the natural antiplasmodial diosgenone / Caracterización parcial del proteoma del trofozoíto de Plasmodium falciparum bajo tratamiento con quinina, mefloquina y el compuesto natural diosgenona

Introduction: Despite efforts to control malaria, around 10% of the world population is at risk of acquiring this disease. Plasmodium falciparum accounts for the majority of severe cases and deaths. Malaria control programs have failed due to the therapeutic failure of first-line antimalarials and to parasite resistance. Thus, new and better therapeutic alternatives are required. Proteomic analysis allows determination of protein expression levels under drug pressure, leading to the identification of new therapeutic drug targets and their mechanisms of action. Objective: The aim of this study was to analyze qualitatively the expression of P.falciparum trophozoite proteins (strain ITG2), after exposure to antimalarial drugs, through a proteomic approach. Materials and methods: In vitro cultured synchronized parasites were treated with quinine, mefloquine and the natural antiplasmodial diosgenone. Protein extracts were prepared and analyzed by two-dimensional electrophoresis. The differentially expressed proteins were selected and identified by MALDI-TOF mass spectrometry. Results: The following proteins were identified among those differentially expressed in the parasite in the presence of the drugs tested: enolase (PF10_0155), calcium-binding protein (PF11_0098), chaperonin (PFL0740c), the host cell invasion protein (PF10_0268) and proteins related to redox processes (MAL8P1.17). These findings are consistent with results of previous studies where the parasite was submitted to pressure with other antimalarial drugs. Conclusion: The observed changes in the P. falciparum trophozoite protein profile induced by antimalarial drugs involved proteins mainly related to the general stress response.

Introducción. A pesar de los esfuerzos para controlar la malaria, esta sigue siendo un problema de salud pública. Plasmodium falciparum es responsable de la mayoría de los casos graves y de las muertes. Los programas de control de la malaria han sido cuestionados debido al fracaso del tratamiento y a la resistencia del parásito a los antipalúdicos de primera línea, por lo que se requieren nuevas y mejores alternativas. El análisis proteómico permite identificar y determinar los niveles de expresión de las proteínas bajo la presión de los medicamentos, lo que posibilita la identificación de nuevos blancos terapéuticos y mecanismos de acción. Objetivo. Analizar cualitativamente la expresión diferencial de proteínas del citosol del trofozoíto de P. falciparum bajo tratamiento con quinina, mefloquina y el compuesto natural diosgenona mediante una aproximación proteómica. Materiales y métodos. Se trataron trofozoítos sincronizados y cultivados in vitro de P. falciparum (cepa ITG2) con quinina, mefloquina y el compuesto natural diosgenona. Los extractos proteicos se prepararon y analizaron por electroforesis bidimensional. Las proteínas con aparente expresión diferencial se seleccionaron e identificaron mediante espectrometría de masas MALDI-TOF. Resultados. Se encontraron las siguientes proteínas diferencialmente expresadas en el trofozoíto: la enolasa (PF10_0155), la proteína de unión a calcio (PF11_0098), la chaperonina (PFL0740c), la proteína de invasión a la célula del huésped (PF10_0268) y la proteína relacionada con procesos de reducción y oxidación (redox) (MAL8P1.17). Estos hallazgos son congruentes con resultados previos de estudios en los que el parásito fue presionado con otros medicamentos antipalúdicos. Conclusión. Los cambios observados en el perfil de proteínas del trofozoíto de P. falciparum tratado con antipalúdicos involucraron preferencialmente proteínas relacionadas con la respuesta al estrés general.

Drug Resistance and in Vitro Susceptibility of Plasmodium falciparum in Thailand during 1988-2003

The aim of the present study was to investigate antimalarial drug pressure resulting from the clinical use of different antimalarials in Thailand. The phenotypic diversity of the susceptibility profiles of antimalarials, i.e., chloroquine (CQ), quinine (QN), mefloquine (MQ), and artesunate (ARS) in Plasmodium falciparum isolates collected during the period from 1988 to 2003 were studied. P. falciparum isolates from infected patients were collected from the Thai-Cambodian border area at different time periods (1988-1989, 1991-1992, and 2003), during which 3 different patterns of drug use had been implemented: MQ + sulphadoxine (S) + pyrimethamine (P), MQ alone and MQ + ARS, respectively. The in vitro drug susceptibilities were investigated using a method based on the incorporation of [3H] hypoxanthine. A total of 50 isolates were tested for susceptibilities to CQ, QN, MQ, and ARS. Of these isolates, 19, 16, and 15 were adapted during the periods 1988-1989, 1991-1993, and 2003, respectively. P. falciparum isolates collected during the 3 periods were resistant to CQ. Sensitivities to MQ declined from 1988 to 2003. In contrast, the parasite was sensitive to QN, and similar sensitivity profile patterns were observed during the 3 time periods. There was a significantly positive but weak correlation between the IC50 values of CQ and QN, as well as between the IC50 values of QN and MQ. Drug pressure has impact on sensitivity of P. falciparum to MQ. A combination therapy of MQ and ARS is being applied to reduce the parasite resistance, and also increasing the efficacy of the drug.

Gametocyte Clearance in Uncomplicated and Severe Plasmodium falciparum Malaria after Artesunate-Mefloquine Treatment in Thailand

Artemisinin-based combination therapy (ACT) is currently promoted as a strategy for treating both uncomplicated and severe falciparum malaria, targeting asexual blood-stage Plasmodium falciparum parasites. However, the effect of ACT on sexual-stage parasites remains controversial. To determine the clearance of sexual-stage P. falciparum parasites from 342 uncomplicated, and 217 severe, adult malaria cases, we reviewed and followed peripheral blood sexualstage parasites for 4 wk after starting ACT. All patients presented with both asexual and sexual stage parasites on admission, and were treated with artesunate-mefloquine as the standard regimen. The results showed that all patients were asymptomatic and negative for asexual forms before discharge from hospital. The percentages of uncomplicated malaria patients positive for gametocytes on days 3, 7, 14, 21, and 28 were 41.5, 13.1, 3.8, 2.0, and 2.0%, while the percentages of gametocyte positive severe malaria patients on days 3, 7, 14, 21, and 28 were 33.6, 8.2, 2.7, 0.9, and 0.9%, respectively. Although all patients were negative for asexual parasites by day 7 after completion of the artesunate-mefloquine course, gametocytemia persisted in some patients. Thus, a gametocytocidal drug, e.g., primaquine, may be useful in combination with an artesunate-mefloquine regimen to clear gametocytes, so blocking transmission more effectively than artesunate alone, in malaria transmission areas.

In vitro study assessing the response of plasmodium falciparum malaria to chloroquine, sulfadoxine/pyrimethamine, quinine and mefloquine in Wad Medani district, Sudan

To assess the in vitro response of Plasmodium falciparum malaria to chloroquine [CQ], sulfadoxine/pyrimethamine [SDX/PYR], Quinine [QU] and Mefloquine [MQ] and monitoring their resistance. In 1999 to 2000, an in vitro study was carried out in Wad Medani district in Sudan. The standard protocol of the WHO in vitro micro-test Mark II was used for the selection of the subjects, the collection of blood samples, the culture techniques, the examination of the post-culture blood slides and the interpretation of the results. In vitro micro-test Mark II were performed on 62 Plasmodium falciparum isolates. Of these isolates, 42 produced successful schizonts growth. The data obtained showed that 29 of 42 isolates [69%] were CQ resistant with an effective concentrations [EC]; EC50 = 399.621 nM, EC90 = 2754.145 nM and EC99 = 13284.967 nM to inhibit the schizont maturation, the values of SDX/PYR showed a flat regression line as an indication of in vitro reduced response with an EC50 = 0.262 nM, EC90 = 147.390 nM and EC99 = 25722.296 nM, and the response to the QU indicated only one of the 42 isolates [2.4%] was resistant with an EC50 = 150.085 nM, EC90 = 822.825 nM and EC99 = 3293.667 nM, while all the 42 isolates were sensitive to MQ with an EC50 = 190.763 nM, EC90 = 615.125 nM and EC99 = 1597.504 nM. The results of this study revealed a high degree of in vitro resistance to CQ and reduced in vitro response to SDX/PYR and QU, while MQ was fully sensitive. The effective concentrations to inhibit 50, 90 and 99% of the parasite maturation were determined for antimalarial drugs efficacy monitoring surveillance in Sudan

In vitro susceptibility and genetic variations for chloroquine and mefloquine in Plasmodium falciparum isolates from Thai-Myanmar border.

In vitro drug susceptibility to chloroquine (CQ) and mefloquine (MF) were assessed in 39 P. falciparum isolates from the Thai-Myanmar border area. To further characterize CQ- and MF-resistance profiles in this area, we also analyzed pfcrt K76T mutation that is critical for CQ resistance, and pfmdr1 polymorphism that has an association with MF resistance. Eighteen isolates were successfully examined by in vitro tests for CQ, and 17 of them had resistance to the drug. Geometric mean concentration of CQ that inhibited the growth of parasites at 50% (IC50) was 371 +/- 227 nM (105-971 nM). Sixteen isolates were successfully examined by in vitro tests for MF, and 8 of them were resistant to the drug. Geometric mean of IC50 for MF was 41 +/- 31 nM (4-125 nM). Genotypes of drug resistance, such as pfcrt and pfmdr1 mutations, were also analyzed. All the 39 isolates had the same haplotype (CVIET) for PfCRT at its 72-76th amino acids. A pfmdr1 Y86 mutation was found in 95% of isolates. A pfmdr1 D1042 mutation was also present in 7 isolates, while no pfmdr1 Y1246 mutation was observed. These results indicated a correlation between CQ resistance and the pfcrt T76 and pfmdr1 Y86 mutations.

Declining mefloquine sensitivity of Plasmodium falciparum along the Thai-Myanmar border.

Mefloquine sensitivity of Plasmodium falciparum along the Thai-Myanmar border, both in vitro and in vivo, following different first-line treatments for uncomplicated falciparum malaria patients in these areas during the period 1997--2003 were studied. Standard in vitro micro tests and in vivo efficacy according to World Health Organization methodologies were performed. P. falciparum isolates along the Thai-Myanmar border with in vitro sensitivity to mefloquine have had up to a ten-fold decrease in sensitivity compared to a baseline done in 1986, conducted one year after the drug was first introduced to Thailand. The reduction in the mefloquine sensitivity of P. falciparum isolates in Tak Province developed rapidly, with the highest IC50 of 1,254 nM in 1997. The IC50 declined to 1,067 and 737 nM in 1999 and 2001, respectively, but there was no statistically significant difference in the sensitivity. The sensitivity of P. falciparum isolates from Mae Hong Son, Kanchanaburi, and Ranong, where the first line treatment was mefloquine 15 mg/kg single dose, continued to decline, where in 2001 the IC50 were 1,087, 941, and 1,116 nM, respectively, in these provinces. The difference in sensitivities of P. falciparum isolates in Mae Hong Son and Ranong in 2001, compared to 1997, was statistically significant (p<0.05). Good therapeutic efficacy of the artesunate-mefloquine combination in Tak Province was observed. Adequate clinical responses (ACR) were 89.5% and 92.3% in 1997 and 2002, respectively. The efficacy of mefloquine alone in Mae Hong Son, Kanchanaburi, and Ranong has significantly dropped. ACR in 1997 and 2001 in Mae Hong Son were 87.8% and 73.2%, respectively, in Kanchanaburi were 82% and 59.6%, respectively, and in Ranong were 96% and 31.6%, respectively.

In vitro susceptibility of Plasmodium falciparum isolates to chloroquine and mefloquine in southeastern Mindanao Island, the Philippines.

Although the presence of multi-drug-resistant falciparum malaria has been reported in the Philippines, the distribution of drug-resistant malaria parasites has not yet been determined in Mindanao Island. In vitro susceptibility of P. falciparum to both chloroquine and mefloquine was assessed to forecast the spread of drug-resistant parasites in various foci in southeastern Mindanao Island. Of the 33 isolates of P. falciparum successfully tested, 10 (30%) were susceptible, 12 (36%) showed decreased susceptibility (80 nM < or = IC50 < 114 nM), and 11 (33%) were resistant (IC50 > or = 114 nM) to chloroquine. Ten (91%) of the resistant isolates and 9 (75%) of those with decreased susceptibility were from northern and northwestern Davao del Norte Province. Chloroquine-susceptible isolates were found among patients in the eastern parts of Davao del Norte and Davao Oriental provinces. Seven isolates from several foci in the study area were all mefloquine- susceptible (IC50 < 10 nM). This is the first report indicating the potential emergence of chloroquine-resistant P. falciparum on Mindanao Island, which is presently regarded as a drug-susceptible area.

Drug resistant malaria on the Thai-Myanmar and Thai-Cambodian borders.

We describe the changing epidemiology of drug resistant malaria in Thailand over the past decade. Factors determining the characteristic patterns of the development and spread of resistance to anti-malarial drugs on the Thai-Cambodian border and the Thai-Myanmar border are explored, namely, population dynamics, drug usage and malaria control measures. The introduction of artesunate-mefloquine combination in selected areas along the two borders in 1995 is believed to be one of the multiple factors responsible for stabilizing the multidrug resistance problems in Thailand today. Other control measures and inter-governmental co-operation must continue to be strengthened in order to limit the spread of drug resistance malaria in the Southeast Asian region.

A simplified in vivo drug sensitivity test for malaria in the field.

The study was intended to develop a simple and reliable in vivo field test for monitoring of sensitivity of P.falciparum to antimalarials. The test is to be used as a built in sustainable monitoring system and applied at regular frequencies to provide guidance in developing a country-wide antimalarial drug policy. The study was conducted as a hospital based study in Mon State in Mudon, Kamawet and Pa-auk hospitals. The criteria matched malaria patients were treated with standard dosages of chloroquine, sulfadoxine-pyrimethamine and mefloquine and blood films were taken on days 0, 2, 3, 4, 7, 14 and 28. The assessment of the in vivo drug response of P.falciparum on days 2, 3 and 4 were compared with WHO standard 28 days and 7 day tests. The following successful tests were carried out for 7 days with different antimalarials: 171 tests with chloroquine and sulfadoxine-pyrimethamine and 167 tests with mefloquine. Tests were also carried out for 28 days: 59 tests with chloroquine, 77 tests with sulfadoxine-pyrimethamine and 78 tests with mefloquine. The results found that 3 day tests, taking blood films on days 0 and 3, can be reliably used as an adjunct to 28-day tests. Since the test is simple and can be used extensively and sustainably throughout the country and the results are applicable to be used for epidemiological purposes, the method is suggested for use as a built-in monitoring method for the malaria control program.

An in vitro study on mefloquine/quinine in Plasmodium falciparum malaria.

An in vitro designed for determining sensitivity of Plasmodium falciparum strains isolated from Kanchanaburi Province, to Mefloquine/Quinine combination, was carried out. The MIC values of Mefloquine/Quinine for the P.falciparum strains were found to be 0.075/3.75 to 0.225/11.15 nM/ml. The changes observed following the drug treatment were an enlargement of the space between the outer and the inner limiting membrane of the parasitophorous vacuole. These changes were followed by cytoplasmic degeneration and vacuolation.