In vitro drug sensitivity testing in Plasmodium vivax.

The possibility of short-term in vitro cultivation, i.e. growth of asexual erythrocytic stages up to the stage of mature schizonts, permits in principle the development of drug sensitivity tests also for Plasmodium vivax. In the absence of a sequestration of erythrocytes carrying the advanced stages of schizogony, asexual parasites of all stages may be seen in the peripheral blood of patients infected with P. vivax. This precludes schizont maturation tests since schizont development will be unduly influenced by the number of advanced trophozoites. A test system reflecting the age composition of the parasite population and its progression without and under the influence of inhibitors was found to yield precise results also in the higher IC ranges. The population-based test procedure would also permit the identification of any stage-specific impact of antimalarial agents.

Characterization of a novel class of antimalarials and its applicability to plasmodial target identification.

Candidate drugs related to the lead compound propafenone are highly effective inhibitors of P. falciparum growth with 50% inhibitory concentrations (IC(50)s) in the sub-micromolar range. The parental compound propafenone is a cardiac sodium channel blocker which is in clinical use for the treatment of ventricular arrhythmia. An in house library of more than 400 compounds with systematically varied structures is available for 2D and 3D quantitative structure activity relationship studies. In a first step selected compounds were evaluated for their antimalarial activity using the histidine-rich protein 2 drug sensitivity assay. Propafenone analogues contain an inherently photoactive aryl-carbonyl substructure, which allows their use in photolabeling studies. Labelling efficiency is increased for compounds in which the phenylpropiophenone core structure is replaced by a benzophenone substructure. However, the phenylpropiophenone substructure represents part of the pharmacophore of the compounds. Benzophenone-type analogues show IC(50) values that are higher than their congeneric phenylpropiophenones. Nevertheless, one of the photoligands shows an IC(50) value in the low micro-molar range. Use of this photoligand is thus expected to allow identification of candidate targets by mass spectrometry following two dimensional separation of the plasmodial proteome. The Malaria Genome Project has advanced our understanding of parasite biology and development of novel drugs can mount on data made available by the recently completed sequencing effort of P. falciparum. The lead compound propafenone is a registered drug and this compound class might therefore have a major potential as an antimalarial drug, either alone, or in combination with conventional antimalarials.

Development of a pharmacodynamic screening model with Entamoeba histolytica.

Human amoebiasis caused by Entamoeba histolytica is widely distributed in the tropics and subtropics, but also occurring in neighbouring parts of the temperate zones. Invasive amoebiasis causes dysentery and, by haematogenous spread, also extra-intestinal hepatic, pulmonary or cerebral abscesses, not rarely fatal conditions. The available anti-amoebic drugs have shortcomings regarding tolerability and efficacy. To facilitate the screening of candidate material, an in vitro system has been developed that permits the determination of specific anti-amoebic activity. PYE medium, supplemented with bovine serum, proved to be suitable for the maintenance of the stock cultures of Entamoeba histolytica strain HM1:1MSS. For sensitivity testing, Waymouth medium and cultivation under aerobic conditions were most reliable. After adapting the system to the use of 96-well (8 x 12) tissue culture plates, sensitivity tests were carried out with metronidazole, dehydroemetine and dihydroartemisinin as active control drugs, and seven extracts from Stemona tuberosa, Aglaia edulis, Aglaia elaeagnoidea and Aglaia odorata. Stem bark extract from Aglaia elaeagnoidea was the most active material with an IC(99) of 496 ng/ml and a slope S of 1.1325, followed by leaf extract from Stemona tuberosa with an IC(99) of 638 ng/ml and a slope S of 1.5648. All seven extracts showed full activity at concentrations <4000 ng/ml and qualified for further investigation.

Clinical-parasitological response and in-vitro sensitivity of Plasmodium vivax to chloroquine and quinine on the western border of Thailand.

This study was conducted during 2002-2004 at Mae Sot District, on the Thai-Myanmar border, an area of multidrug-resistant Plasmodium falciparum malaria. Sixty-two patients with P. vivax malaria were included in the study. All were randomized into two groups to receive a 3-day regimen of chloroquine or a 3-day regimen of quinine. Primaquine was given to patients in both groups for the elimination of hepatic stages. Results from the present study suggest that the standard regimen of chloroquine and a 3-day course of quinine at the dose regimens under investigation were very effective and well tolerated for the treatment of P. vivax malaria in this area. All patients responded well to both drug regimens; the cure rates with chloroquine or quinine, when given concurrently with the tissue schizontocidal drug primaquine, were virtually 100% within 28 days of follow-up. No significant correlations between parasite clearance time (PCT) or fever clearance time (FCT) and inhibitory concentration 50 (IC50) were found. Patients who had PCT < or = 24 h and those with PCT >24 h had comparable IC50 to chloroquine (alone and plus primaquine) and quinine, as well as similar concentrations of chloroquine/desethylchloroquine (in blood) or quinine (in plasma) at the investigated time points.

Development of a pharmacodynamic screening model with Crithidia fasciculata.

The genus Crithidia is a member of the family Trypanosomatidae and is related to the genera Leishmania and Trypanosoma with which it shares a variety of biochemical mechanisms, such as polyamine synthesis and methionin salvage. In consequence, a screening system for antiparasitic candidate material has been developed with Crithidia fasciculata, a parasite naturally occurring in insects and amphibians, but devoid of pathogenicity for humans. Initially a variety of culture media were evaluated of which TPS was best suited for the maintenance of stock cultures, and E-medium - a newly developed formula - for sensitivity testing. Optimal growth of C. fasciculata was observed under microaerophilic conditions. A system for sensitivity testing was developed and applied to the investigation of extracts from higher tropical plants of the genera Stemona and Aglaia for anticrithidial activity. Extracts with significant anti-crithidial activity were scheduled for chromatographic fractionation and the subsequent isolation, purification and structural identification of individual compounds for further sensitivity testing. Encouraging results were obtained with extracts from Aglaia odorata leaves, A. elaeagnoidea stem bark and A. edulis leaves, with EC(90) values of 1213 ng/ml, 1606 ng/ml, and 1462 ng/ml, respectively.

An in vitro system for assessing the sensitivity of Plasmodium vivax to chloroquine.

Following earlier observations on short-term culture of Plasmodium vivax, an in vitro test system has been developed for assessing the parasite's sensitivity to chloroquine. Fresh isolates with predominantly young trophozoites are diluted 1:19 with a (v/v=1/1) mixture of RPMI 1640 and Waymouth medium. The blood-medium mixture (BMM) is inoculated into the predosed microtitre plates before incubation in a candle jar. Incubation for 30 or 42 h yielded the best results. Incubation for 18 or 24 h was generally insufficient for an adequate development of the parasites. The reading of the test is based on stage-specific differential counts in the Giemsa-stained pre-incubation and post-incubation thick films, the evaluation on log-probit analysis of drug-related inhibition of parasite development. The test system has been evaluated on 200 fresh P. vivax isolates in an area with satisfactory clinical-parasitological response to chloroquine. At 30 or 42 h incubation 121 isolates (61.5%) showed adequate control growth and yielded valid sensitivity tests. Complete inhibition of parasite development occurred within the concentration range of 40-1280 nM. The mean EC50 for 30 h of incubation was 50.3 nM, as compared to 49.7 nM with 42 h of incubation. The geometric mean cut-off concentration of parasite development was 488 nM with 30 h of incubation as against 470 nM with 42 h of incubation.