Activity of selected phytochemicals against Plasmodium falciparum.

According to the WHO, in 2008, there were 247 million reported cases of malaria and nearly one million deaths from the disease. Parasite resistance against first-line drugs, including artemisinin and mefloquine, is increasing. In this study the plant-derived compounds aglafolin, rocaglamid, kokusaginine, arborine, arborinine and tuberostemonine were investigated for their anti-plasmodial activity in vitro. Fresh Plasmodium falciparum isolates were taken from patients in the area of Mae Sot, north-western Thailand in 2008 and the inhibition of schizont maturation was determined for the respective compounds. With inhibitory concentrations effecting 50%, 90% and 99% inhibition (IC(50), IC(90) and IC(99)) of 60.95 nM, 854.41 nM and 7351.49 nM, respectively, rocaglamid was the most active of the substances, closely followed by aglafoline with 53.49 nM, 864.55 nM and 8354.20 nM. The activity was significantly below that of artemisinin, but moderately higher than that of quinine. Arborine, arborinine, tuberostemonine and kokusaginine showed only marginal activity against P. falciparum characterized by IC(50) and IC(99) values higher than 350 nM and 180 µM, respectively, and regressions with relatively shallow slopes S>14.38. Analogues of rocaglamid and aglafoline merit further exploration of their anti-plasmodial activity.

Sensitivity of Plasmodium vivax to chloroquine, mefloquine, artemisinin and atovaquone in north-western Thailand.

Excepting tropical Africa, where Plasmodium falciparum prevails, Plasmodium vivax is the most frequent cause of malaria in Asia and Latin America. First reliable reports of chloroquine resistance came in 1989 from the area of the distribution of the Chesson-strain of P. vivax. Since then, reports also came from other areas of the world. This study had the objective of measuring the sensitivity of P.vivax to chloroquine and potential alternative compounds in western Thailand. The study was performed in 2008 in Mae Sot, Tak Province, and followed the method of Tasanor. The IC(50) and IC(90) values for chloroquine were 167 nM and 5445 nM, those for mefloquine were 139 nM and 5282 nM, those for artemisinin were 32 nM and 466 nM, and those for atovaquone 30 nM and 650 nM. The values for chloroquine indicate the existing or imminent occurrence of specific resistance. High prevalence of mefloquine resistance precludes its alternative use. However, atovaquone, in combination with proguanil, may be a possible alternative.

Pharmacodynamic interaction between lumefantrine and desbutyl-benflumetol in Plasmodium falciparum in vitro.

The pharmacodynamic interaction between lumefantrine and monodesbutyl-benflumetol has been investigated in 44 fresh isolates of patients with a Plasmodium falciparum infection from the region of Mae Sot (Thailand). Both substances proved to be effective against parasite maturation within the test concentration range, with monodesbutyl-benflumetol being effective at a lower concentration than lumefantrine. Synergism between the two substances was evaluated with a combination of lumefantrine and monodesbutyl-benflumetol at a ratio of 4.25:1. The geometric mean values for complete inhibition of schizont maturation were 1035.7 nM for lumefantrine, 655 nM for monodesbutyl-benflumetol and 222.5 nM for the combination of both. An analysis for interaction according to the method of Berenbaum indicates a moderate synergism at the IC(50), which gets stronger with increasing ICs and reaches the highest level at the IC(99). The geometric mean of the sums of the FIC(50) is 0.73, of the FIC(90) it is 0.37 and of the FIC(99) it is 0.25.

Synergism between pyronaridine and retinol in Plasmodium vivax in vitro.

Estimates of the annual number of infections with Plasmodium vivax reach 391 million. So far the blood-schizontocidal therapy with chloroquine remained effective in most parts of the world, but reports about emerging resistance are increasing. The study had the objective of determining the pharmacodynamic interaction between pyronaridine and retinol in Plasmodium vivax, since pyronaridine is a potential alternative for chloroquine and an earlier study had shown strong synergism between pyronaridine and retinol in Plasmodium falciparum. The study was conducted at the Malaria Clinic of Mae Sot, Tak Province, Thailand, near the border to Myanmar. The in vitro observations followed the method of Tasanor. Successful tests were performed with 44 isolates. The mean IC(50), IC(90) and IC(99) values for pyronaridine were 9.8, 2069.6 and 162446.5 nM. The mean IC(50), IC(90) and IC(99) values for the combinations with retinol (corresponding to the 50th, 65th and 80th percentile of the physiological retinol levels in healthy adults) were 1.7, 542.8 and 59379.5 nM for pyronaridine + retinol "low", for the combination with retinol "medium" they were 0.5, 313.7 and 58891.4 nM and for the combination with retinol "high" they were 0.2, 96.7 and 16754.3 nM. These results suggest strong synergism between the two substances.

Synergism between mefloquine and artemisinin and its enhancement by retinol in Plasmodium falciparum in vitro.

Following the advent of mefloquine resistance in Plasmodium falciparum in Thailand in the 1990s, the combined treatment of falciparum malaria with artesunate and mefloquine was found to be highly effective in treating and curing the patients in the affected areas. Monitoring of the clinical-parasitological response and of the in vitro sensitivity of P. falciparum was systematically conducted in order to detect any signs of failure of this type of artemisinin-based combination treatment (ACT). In earlier observations the in vitro activity of artemisinin was found to be significantly enhanced when combined with retinol. The same applies to mefloquine. In order to check whether the synergism between artemisinin and mefloquine was maintained in the presence of retinol, the pharmacodynamic interaction of the three compounds was investigated in the western border area of Thailand. Successful parallel tests with mefloquine, artemisinin, retinol, mefloquine-artemisinin 5:1 as well as mefloquine-artemisinin (5:1) + retinol low, medium and high were obtained with 43 fresh parasite isolates. The retinol concentrations in the low, medium and high formulations corresponded to the 50th, 65th and 80th percentile of the physiological mean concentrations in the blood of healthy adults. The IC(50), IC(90) and IC(99) values for mefloquine alone showed a further increase over the data of 2008. In the combinations with artemisinin and retinol moderate synergism was observed at the IC(50), but synergism increased strongly at the IC(90) and the IC(99).

Sensitivity to artemisinin, mefloquine and quinine of Plasmodium falciparum in northwestern Thailand.

The increasing drug resistance of Plasmodium falciparum is a worldwide problem. The objective of the study was the assessment of the in vitro activity of artemisinin, mefloquine and quinine, in an area where P. falciparum is multi-drug resistant. The sensitivity tests were based on measuring the drug-dependent inhibition of schizont maturation. For the 43 successfully tested isolates the mean effective concentrations (IC(50) and IC(90)) for artemisinin were 0.0081 and 0.1372 µM, respectively. For mefloquine the IC(50) was 0.1260 µM and the IC(90) was 3.7345 µM. Quinine showed an IC(50) of 0.2155 µM and an IC(90) of 2.5040 µM. All tested drugs showed a significant reduction in the effectiveness, compared with the results of former years. This suggests a further rise of resistance of local P. falciparum, which is alarming especially for artemisinin and quinine.

Activity of Eurycoma longifolia root extract against Plasmodium falciparum in vitro.

The habitats of Eurycoma longifolia Jack, a slender tree, are jungles in Malaysia and Indonesia. It belongs to the family Simaroubaceae and is a source of quassinoids with anabolic, antimalarial and cytostatic activity. In this study, conducted during 2008 in Mae Sot, Thailand, a standardized extract of E. longifolia containing three major quassinoids, eurycomanone (1), 13,21-dihydroeurycomanone (2) and 13alpha(21)-epoxyeurycomanone (3) was evaluated for antiplasmodial activity against Plasmodium falciparum and its activity has been compared with that of artemisinin, using 38 fresh parasite isolates and assessment of inhibition of schizont maturation. The IC(50), IC(90) and IC(99) values for artemisinin were 4.30, 45.48 and 310.97 microg/l, and those for the root extract from E. longifolia 14.72, 139.65 and 874.15 microg/l respectively. The GMCOC for artemisinin was 337.81 mug/l, and for the plant extract it was 807.41 microg/l. The log-concentration probit regressions were parallel. The inhibitory activity of the E. longifolia extract was higher than that expected from the three quassinoids isolated from the plant, suggesting synergism between the quassinoids or the presence of other unidentified compounds.

Pharmacodynamic interaction between mefloquine and retinol in Plasmodium falciparum in vitro.

Mefloquine, a 4-quinolinemethanol derivative, was introduced in Thailand after Plasmodium falciparum had acquired almost universal resistance to the 4-aminoquinolines and antifols. However, also resistance to mefloquine has become an increasing problem, but artemisinin-based combination therapy (ACT) with mefloquine and artesunate remained until recently sufficiently effective. Since synergistic interaction between quinine, another 4-quinolinemethanol, with retinol was observed earlier, the investigations were expanded to mefloquine. The interaction between mefloquine and retinol at concentrations equal to the 50(th), 65(th) and 80(th) percentile of the physiological retinol levels in healthy adults was determined in 37 fresh isolates of P. falciparum. The mean IC(50), IC(90) and IC(99) values for mefloquine were 1.76, 9.81 and 39.78 microM, those for mefloquine + retinol "low" 0.33, 1.37 and 4.33 microM, those for mefloquine + retinol "medium" 0.29, 1.15 and 3.48 microM, and those for mefloquine + retinol "high" 0.20, 0.85 and 2.70 microM. Evidence for strong synergism between mefloquine and retinol in P. falciparum was highly significant.

Synergism between pyronaridine and retinol in Plasmodium falciparum in vitro.

Pyronaridine, a naphthyridine derivative and Mannich base, is a highly active blood schizontocide and currently being explored as partner in artemisinin-based combination therapy (ACT). In this study, carried out 2008 in Mae Sot, Thailand, the activity of pyronaridine was found to be compromised, obviously as the result of mono-therapy in an adjacent area. The pyronaridine sensitivity and the interaction between pyronaridine and retinol at concentrations corresponding to the 50th, 65th and 80th percentile of the physiological retinol levels in healthy adults were assessed in 38 fresh isolates of P. falciparum. The mean IC50, IC90 and IC99 values for pyronaridine were 12.7, 201.4 and 3084.2 nM, those for pyronaridine + retinol "low" 1.2, 14.0 and 102.6 nM, those for pyronaridine + retinol "medium" 0.6, 7.4 and 54.8 nM, and those for pyronaridine + retinol "high" 0.9, 8.2 and 47.8 nM. There was significant evidence of strong synergism between pyronaridine and retinol against P. falciparum.

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.

Malariological baseline survey and in vitro antimalarial drug resistance in Gulu district, Northern Uganda.

A comprehensive, representative malaria survey has been carried out in a population of internally displaced persons (IDP) in the district of Gulu, Northern Uganda. It included 74 households and 390 persons, and covered socio-economic and environmental information, individual physical data, malaria and the drug sensitivity of Plasmodium falciparum. The prevalence of infections with Plasmodium falciparum was 54.4% at a geometric mean asexual parasitaemia of 229/microl blood, typical for hyperendemic conditions. P. falciparum turned out to be highly resistant to chloroquine and amodiaquine. It showed also reduced sensitivity against lumefantrine and artemisinin, obviously the result of the liberal use of the lumefantrine-artemether combination without evidence-based indication.

Pharmacodynamic interaction between 4-aminoquinolines and retinol in Plasmodium falciparum in vitro.

The pharmacodynamic interaction between retinol and 4-aminoquinolines has been investigated in 29 fresh isolates of Plasmodium falciparum. Although the parasites were highly resistant against 4-aminoquinolines, significant synergism was observed between chloroquine and retinol as well as amodiaquine and retinol, the latter at physiological concentrations. Combination with retinol reduced the geometric mean concentrations effecting complete inhibition (GMCOC) by chloroquine from 14425 nM to 8943 nM in CHL-RET low, 7042 nM in CHL-RET medium, and 4920 nM in CHL-RET high. Synergism between amodiaquine and retinol was greater, with strong and highly significant reductions of the GMCOC, from 2520 nM for amodiaquine to 1092 nM for AMO-RET low, 800 nM for AMO-RET medium, and 745 nM for AMO-RET high. While it is obviously too late for making practical use of the activity enhancement for chloroquine, the situation is different for amodiaquine, where supplementation with retinol may extend the usefulness of the medicament.

Synergism between quinine and retinol in fresh isolates of Plasmodium falciparum.

Following earlier reports of synergism between retinol and various antimalarial compounds, the pharmacodynamic interaction between retinol and quinine was investigated in 38 fresh isolates of Plasmodium falciparum. The study was carried out in western Thailand, an area with quinine-resistant P. falciparum. The combination of quinine with retinol in concentrations corresponding to the 50(th), 65(th) and 80(th) percentile of the physiological values in healthy subjects, significantly reduced the EC(50), EC(90), EC(99) and GMCOC for quinine. The FIC values at EC(90) and EC(99) indicate increasing synergism with rising EC and retinol concentration. The mean SFIC value dropped to a level as low as 0.2420, indicating strong synergism.

Synergism between monodesbutyl-benflumetol and artemisinin in Plasmodium falciparum in vitro.

The sensitivity to artemisinin, monodesbutyl-benflumetol (DBB) and a 1:1 m/m combination of the two compounds was successfully investigated on 34 fresh isolates of Plasmodium falciparum. On a molar basis the combination was most active, followed by DBB and artemisinin. The geometric mean concentrations effecting full inhibition (GMCOC) were 49.25 nM for the combination, 279.12 nM for DBB, and 494.05 for artemisinin. The difference between the efficacy of the combination and that of its components was highly significant. Interaction between artemisinin and DBB showed moderate synergism at the EC(50) and strong synergism at EC(90) and EC(99). The individual parasite isolates showed a significant inverse correlation between the ECs and the degree of synergism. Positive specific pharmacodynamic interaction was therefore most marked in isolates with reduced sensitivity against artemisinin and DBB.

Interaction between lumefantrine and monodesbutyl-benflumetol in Plasmodium falciparum in vitro.

The pharmacodynamic interaction between lumefantrine and its monodesbutyl analogue (DBB) has been investigated in 35 fresh isolates of Plasmodium falciparum. Both compounds showed highly significant activity correlation. The geometric mean values for complete inhibition of schizont maturation (GMCOC) were 536,8 nM for lumefantrine, 246.0 nM for DBB, 235,5 nM for LUM-DBB 999:1, and 155,2 nM for LUM-DBB 995:5, with significant activity differences between lumefantrine and DBB as well as the LUM-DBB combinations. For the combination of lumefantrine and DBB 995:5 the sums of the fractional inhibitory concentrations according to Berenbaum (SFIC) indicated marked synergism, the intensity of interaction rising with the effective inhibitory concentrations.

Pharmacodynamic interaction between monodesbutyl-benflumetol and artemisinin as well as proguanil in Plasmodium falciparum in vitro.

The sensitivity of Plasmodium falciparum against artemisinin, monodebutyl-benflumetol (DBB) and a 1:3 m/m combination of both compounds was assessed in 51 fresh parasite isolates. Although a comparison between fully inhibitory concentrations (GMCOC) of artemisinin alone (63.33 nM), DBB alone (50.15 nM) and the combination (23.92 nM) indicated significant synergism between artemisinin and DBB, this was less evident when comparing the log-probit regressions. Moreover, the geometric mean values of the fractional inhibitory concentrations (SFIC) showed a rising tendency with increasing EC level. In a study comprising 24 fresh isolates of P. falciparum, the interaction between DBB and proguanil was explored with a 3:1 m/m combination of both compounds. Proguanil alone showed weak blood schizontocidal activity. The log-probit regressions indicated higher activity of the combination as compared to DBB alone. The SFIC values indicated moderate synergism between DBB and proguanil that could be an advantage in an eventual therapeutic and prophylactic use of DBB.

Synergistic interaction between atovaquone and retinol in Plasmodium falciparum in vitro.

The study has been conducted with the objective of assessing the blood schizontocidal activities of atovaquone (ATO), retinol (RET) and combinations of both (ATO-RET) at set retinol concentrations corresponding to the 50th, 65th and 80th percentile of the physiological serum retinol levels. The in vitro tests followed the WHO standard protocol Mark II for measuring the inhibition of schizont maturation in Plasmodium falciparum. Valid results for all 5 test lines were obtained with 26 fresh parasite isolates from northwestern Thailand, an area affected by multidrug-resistance. The EC(50) values for atovaquone, retinol and for ATO in ATO-RET low, medium and high were 3.1 nM, 561.8nM, 0.85 nM, 0.73 nM and 0.45 nM, respectively, the EC(90) values 33.7 nM, 9338.6 nM, 25.31 nM, 8.89 nM, and 5.42 nM. The geometric mean cut-off concentrations of schizont maturation of atovaquone alone and for atovaquone in ATO-RET low, medium and high were 282.5 nM, 79.0 nM, 38.7 nM and 23.7 nM, respectively. These results and those of the Berenbaum analysis based on the fractional inhibitory concentrations indicate synergistic pharmacodynamic interaction between atovaquone and retinol, a phenomenon suggesting that the antimalarial activity of atovaquone could be enhanced by supplementation with retinol.

Synergistic interaction between monodesbutyl-benflumetol and retinol in Plasmodium falciparum.

The blood schizontocidal activity of monodesbutyl-benflumetol (DBB), retinol (RET) and combinations (DBB-RET) at retinol concentrations corresponding to the 50th, 65th and 80th percentile of physiological retinol concentrations in healthy adults has been investigated in Plasmodium falciparum. Parallel in vitro tests with DBB, RET and the 3 DBB-RET combinations were carried out with 26 fresh parasite isolates from northwestern Thailand, following the WHO standard protocol Mark II for determining the inhibition of schizont maturation. The EC(50) values for DBB, RET and for DBB in DBB-RET low, medium and high were 5.72 nM, 561.83 nM, 1.68 nM, 0.60 nM and 0.07 nM, respectively, the EC(90) values 44.14 nM, 9338.60 nM, 49.00 nM, 28.48 nM and 8.94 nM. The geometric mean cut-off concentrations of schizont maturation for DBB alone and for DBB in DBB-RET low, medium and high were 153.20 nM, 62.93nM, 34.00 nM and 13.74 nM, respectively, indicating significant synergistic interaction between DBB and retinol. The degree of synergism increases with the retinol concentration in the combination and is highest at the EC(99) level for DBB.

Specific pharmacokinetic interaction between lumefantrine and monodesbutyl-benflumetol in Plasmodium falciparum.

The blood schizontocidal activity of lumefantrine, monodesbutyl-benflumetol (DBB) and a 999:1 combination of both compounds has been investigated in 26 fresh isolates of Plasmodium falciparum from northwestern Thailand, using the WHO standard protocol Mark II for determining the inhibition of schizont maturation. The geometric mean cut-off concentrations of schizont maturation (GMCOC) were 943.2 nM for lumefantrine, 146.3 nM for DBB and 182.2 nM for the 999:1 combination of lumefantrine and DBB. The EC(50) values were 27.3 nM for lumefantrine, 5.7 nM for DBB, and 16.5 nM for the combination, and the EC(90) values 163.1 nM for lumefantrine, 44.1 nM for DBB, and 78.3 nM for the combination. Despite the very low concentration in the combination, DBB exerted significant synergistic activity with lumefantrine that was strongest at the EC(90) and EC(99) levels. Correlation analysis indicates that DBB is the leading determinant for the activity of the combination.

Susceptibility to chloroquine, mefloquine and artemisinin of Plasmodium vivax in northwestern Thailand.

The in vitro study had the objectives of monitoring the sensitivity of Plasmodium vivax to chloroquine and artemisinin, and to assess its baseline sensitivity to mefloquine in northwestern Thailand in an area near the border to Myanmar. The investigations were carried out in 2004 at the malaria clinics of Mae Sot, Chedi Ko and Mae Ka Sa, all in the district of Mae Sot, Province of Tak. The in vitro tests followed the method of Tasanor. Successful tests were obtained with 45 fresh isolates of P. vivax. The EC(50) and EC(90) values for chloroquine were 120.9 nM and 655.7 nM, respectively, the GMCOC was 1699.7 nM. There was a significant decrease of the chloroquine sensitivity since 1998/1999. However, results of parallel investigations continue to indicate clinical-parasitological sensitivity to chloroquine. With mefloquine the EC(50) and EC(90) the (baseline) values were 131.6 nM and 972.6 nM, respectively, the GMCOC was 1987.0 nM. For artemisinin the EC(50) and EC(90) values were 8.7 nM and 105.2 nM, respectively, the GMCOC was 310.5 nM. As compared to 2002, the sensitivity of P. vivax to artemisinin has shown a slight but not significant increase.