Therapeutic efficacy of artesunate in Plasmodium vivax malaria in Thailand.

Our previous study showed that in vitro susceptibility of Plasmodium vivax to chloroquine has significantly decreased in Thailand within the past two decades. Thus, the evaluation of alternative antimalarials for treatment of vivax malaria is needed. The aim of this study was to examine parasitological and clinical efficacy of an artemisinin derivative (artesunate) for the treatment of vivax malaria in patients who were admitted to the Bangkok Hospital for Tropical Diseases. We randomly allocated patients aged 12-56 years to receive 3.3mg/kg (adult dose 200 mg) on the first day, and for the next four days each patient was given 1.65 mg/kg orally (adult dose 100 mg), total dose = 600 mg. After the five-day course of artesunate, primaquine was given: a single oral dose of 15mg for 14 days. A total number of 42 patients received treatment. All participants were followed up for 28 days. In all the cases, both parasitemia and fever were resolved rapidly; the mean fever clearance time and parasite clearance time, 14.6 and 36.7 hours, respectively, showed that therapeutic response to artesunate was better than that of chloroquine. The 14-day cure rate was 100%, but reappearance of parasitemia was seen in two patients on days 21 and 25 following treatment, respectively. These two cases of failure rate should be considered as true relapse rather than recrudescence, since the relapse interval in Southeast Asian vivax malaria according to recent findings seems to be 3 weeks after start of treatment, if primaquine is not given or an inadequate amount is given. In conclusion, artesunate might be useful in treatment of vivax malaria, causing a good blood schizontocidal effect. However, to prevent emerging resistance it should never be used alone.

Plasmodium vivax malaria in Southeast Iran in 1999-2001: establishing the response to chloroquine in vitro and in vivo.

Chloroquine-resistant Plasmodium vivax is emerging in Oceania, Asia and Latin America. The drug sensitivity of P. vivax to chloroquine both in vivo and in vitro in the southern part of Iran was assessed; chloroquine-resistant Plasmodium falciparum has already been documented in this area. The in vitro sensitivity of 39 P. vivax isolates was assessed: the mean IC50 and IC90 were 189 ng/ml and 698 ng/ml blood respectively; for in vivo testing, all 39 vivax malaria patients were treated with a standard regimen of chloroquine and followed-up at 28 days: the mean parasite clearance time was 67.2 +/- 22.5 hours. The in vitro development of young parasites to mature schizonts in standard test medium was compared with that obtained in McCoy's 5A medium: no significant difference was observed. Synchronization of the blood-stage parasites was performed according to Lambros' method: the method was not suitable because it was detrimental to the parasites. A number of in vitro tests were performed using both our own laboratory-predosed microplates and WHO microplates: there was no significant difference between the results.

Ex vivo blood schizontocidal activities of artemisinin derivatives against Plasmodium falciparum.

Serum samples collected at intervals from eight healthy volunteers after the administration of the six regimens of artemisinin derivatives were investigated for their ex vivo blood schizontocidal activities against K1 strain Plasmodium falciparum. The regimens included single doses of (a) 300 mg oral artemether; (b) 300 mg intramuscular artemether; (c) 100 mg suppository artemether; (d) 300 mg oral artesunate (Guillin formulation); (e) 300 mg oral artesunate (Arenco formulation); (f) 300 mg oral dihydroartemisinin. Sera collected after various regimens of artemisinin derivatives showed distinct degree of ex vivo blood schizontocidal activities. Activity of sera after suppository dosing was remarkably low and variable comparing to the other two formulations (oral, intramuscular). Median values for Amax (the maximum activity normalized with dose) of sera from oral dosing were 2.4- and 118-fold, while AUA (the area under activity-time curve, normalized with dose) were 0.82- and 2,370-fold of that after the intramuscular and suppository dosing, respectively. Sera from artesunate-Arenco dosing exhibited significantly higher Amax and AUA (medians: Amax 12.4 vs 5.13 nmol/l/mg dose; AUA: 21.9 vs 8.8 nmol x h/ml/mg dose), compared to that from artesunate-Guillin dosing. Among the oral formulations of artemisinin derivatives investigated (artemether, artesunate, dihydroartemisinin), sera collected following a single dose of oral dihydroartemisinin exhibited lowest bioactivity (Amax 2.35 nmol/l/mg dose; AUA: 44 nmol x h/ml/mg dose).

Plasma containing artemether-pyrimethamine has ex vivo blood schizonticidal activity against Plasmodium falciparum.

Plasma samples collected at intervals from healthy volunteers, after administration of 3 drug regimens [artemether (ART) 300 mg, pyrimethamine (PYR) 100 mg, and ART 300 mg plus PYR 100 mg] were examined for blood schizonticidal activity against K1 strain and T(9/94) clone of Plasmodium falciparum ex vivo. A synergistic effect against T(9/94), a pyrimethamine sensitive clone, was observed in plasma collected after ART+PYR administration, when the test was carried out in low p-aminobenzoic acid, low folic acid medium. The maximum activity (Amax), expressed as equivalent dihydroartemisinin concentration, for plasma samples collected after the combined ART+PYR regimen [6,935 (1,330-13,400) nmol/l] was significantly higher than those for the single ART or PYR regimens [935 (397-2,000) and 9.9 (5.6-15.6) nmol/l, respectively]. In addition, the area under the activity curve (AUA) for the combined regimen [12,8397 (39,274-19,7901) nmol.h/l] was significantly higher than those for the single ART or PYR regimens [(3618 (1406-5597) or 334 (82.3-733.3) nmol.h/l, respectively]. Microscopic observation revealed that ART in the combined regimen exerted its inhibitory effect against all erythrocytic stages and that this occurred before effects of PYR activity. Prolongation of inhibitory effects for the combined ART+PYR regimen was shown to be due to PYR activity by comparison to the activity from the single ART regimen. Results clearly demonstrated no PYR activity against K1, a pyrimethamine resistant strain, in plasma samples collected after the single PYR regimen and the ART+PYR regimen. Microscopic examination confirmed that growth inhibition of K1 was caused by ART activity only.

Pharmacokinetic interactions of artemether and pyrimethamine in healthy male Thais.

The pharmacokinetics of a single oral dose of artemether (300 mg) and pyrimethamine (100 mg) given as each individual drug alone or as a drug combination (artemether 300 mg plus pyrimethamine 100 mg), were investigated in 8 healthy male Thai volunteers. Both artemether and pyrimethamine were rapidly absorbed after oral administration. Elimination of pyrimethamine was however, a relatively slow process compared with artemether, and thus resulted in a long terminal phase elimination half-life (50-106 hours). Pharmacokinetics of artemether and dihydroartemisinin following a single oral dose of artemether alone or in combination with pyrimethamine were similar. In contrast, coadministration of artemether resulted in significantly increased Cmax (medians of 818 vs 1,180 ng/ml) and contracted the apparent volume of distribution (medians of 3 vs 2.56 l/kg) of pyrimethamine.

Artemether-pyrimethamine in the treatment of pyrimethamine-resistant falciparum malaria.

In vitro susceptibility and clinical response of multidrug resistant Plasmodium falciparum to the combination artemether-pyrimethamine were evaluated in patients with acute uncomplicated falciparum malaria. Sixty patients were randomized to receive 3 oral regimens of the combination artemether-pyrimethamine as follows: Regimen-I: artemether (300 mg) plus pyrimethamine (100 mg) on the first day, then placebo on the two consecutive days; Regimen-II: artemether (300 mg) plus pyrimethamine (100 mg) on the first day, then artemether (150 mg) plus pyrimethamine (50 mg) on the second day, and placebo on the third day; Regimen-III: artemether (300 mg) plus pyrimethamine (100 mg) on the first day, then artemether (150 mg) plus pyrimethamine (50 mg) on the second and third days. All patients had a rapid initial response to treatments with 95% of parasitemia being cleared within the first 24 hours. PCT24hours and PCT48hours were similar among the three drug regimens (11 vs 4, 6 vs 12, and 9 vs 11 patients for a 1-day, 2-day, and 3-day combination regimen, respectively). Fever was cleared within 48 hours in all patients in either group. Transient mild nausea, vomiting and loss of appetite were found in a few patients during the first 2 days of treatment. Seven patients did not complete the 28 day follow-up period (5 vs 2 in a 1-day vs 2-day regimen), the reason for withdrawal was not associated with drug-related adverse effects. Only 53 patients were therefore qualified for the efficacy assessment. There was 15, 13 and 5 patients in a 1-day, 2-day and 3-day combination regimens, respectively, who had reappearance of the parasitemia between days 11 and 21. The cure rates of the 3 treatment groups were statistically significantly different (0, 27.8, and 75% for a 1-day, 2-day and 3-day combination regimen, respectively). Two patients developed P. vivax malaria on days 20 and 24. All of the isolates were highly resistant to pyrimethamine, with MIC of 10(-5) M. There is potential advantage of this combination therapy in reducing the dosage and treatment period of artemisinin derivative, which is therefore likely to improve complaince in clinical practice. The use of a 3-day combination regimen (300 mg artemether plus 100 mg pyrimethamine on the first day, then 150 mg artemether plus 50 mg pyrimethamine on the second and third days) seems to be a good alternative regimen to sulfadoxine/ pyrimethamine in areas where P. falciparum is sensitive to pyrimethamine eg in Africa.

Development of in vitro microtest for the assessment of Plasmodium vivax sensitivity to chloroquine.

An in vitro microtest to assess P. vivax sensitivity to chloroquine has been developed using a medium mixture which contained RPMI, Waymouth (GIBCO) and 15% (vol/vol) human serum group AB. The rate of success was highest in samples which were not washed by centrifugation before culturing in microtest plates predosed with varying concentrations of chloroquine. Evaluation of the effective concentration of chloroquine using a program for probit analysis of log dose/response test proved superior to simply using the percentage of schizont maturation.

Flunarizine and verapamil inhibit chloroquine-resistant Plasmodium falciparum growth in vitro.

Using pharmacological properties in relation to the biochemistry of P. falciparum, verapamil, flunarizine, and chlorpromazine which are calcium blockers were selected to test for their antimalarial activity against P. falciparum in vitro. Results revealed that the drugs inhibited parasite population growth in the following order of IC50: verapamil 1 X 10(-6) M, chlorpromazine 3.5 X 10(-6) M, and flunarizine 5 X 10(-6) M. These three calcium blockers have antimalarial effects on chloroquine resistant parasite (alone T9/94) but are less potent when compared with the efficacy of quinine or mefloquine in vitro.

Mefloquine-pyrimethamine-sulfadoxine combination delays emergence of mefloquine resistant Plasmodium falciparum in continuous culture.

The efficacy of mefloquine against Plasmodium falciparum in continuous culture was studied. The development of mefloquine resistance in P. falciparum was significantly inhibited by a combination of mefloquine, pyrimethamine and sulfadoxine. By contrast, more resistant variants were selected in continuous culture without drug pressure or with pyrimethamine-sulfadoxine pressure. The most mefloquine resistant variants were selected by step-wise increases in mefloquine pressure.

Cross resistance of pyrimethamine and sulfadoxine to their related compounds in Plasmodium falciparum.

Cross resistance of pyrimethamine and amethopterin, sulfadoxine and the other sulfonamides in Plasmodium falciparum culture lines was studied. Our results indicate some evidence of a cross resistance between pyrimethamine to amethopterin a drug sharing the same mode of action but never been used as an antimalarial before. Studies on sulfonamides revealed that the minimal inhibitory concentration for sulfadoxine was lower than for sulfadiazine and sulfisoxasole, and that a cross resistance between sulfadoxine and the other sulfonamides may not occur.