Investigations of incidence of pretreatment, drug sensitivity in vitro, and plasma levels of pyrimethamine in patients with multidrug resistant falciparum malaria following the three combination regimens of artemether/pyrimethamine

The study was carried out to investigate the status of in vitro susceptibility of Plasmodium falciparum to pyrimethamine (PYR) in multidrug resistant area of the Thai-Myanmar border, the incidence of unregulated use of the combination of PYR with sulfadoxine (Fansidar) in this area and the relevance of pharmacodynamic and pharmacokinetic factors in determining the treatment outcome from the three combination regimens of ART/PYR (1-, 2- and 3-day regimens), in patients with acute uncomplicated falciparum malaria. The majority of patients had baseline PYR concentrations in the range of 1-100 (50.6%) or 100-500 (34.8%) ng/ml, while concentrations of more than 500 ng/ml were found in only 1.1%. All of the isolates exhibited high grade resistance to PYR with the minimum inhibition concentration (MIC) of as high as 10(-5) M. No association was observed between treatment outcome and the presence of baseline plasma PYR concentrations. In addition, lack of association between plasma concentrations during the acute phase (day-1 and -2) and treatment outcome was found.     

Associations between frequencies of a susceptible TNF-alpha promoter allele and protective alpha-thalassaemias and malaria parasite incidence in Vanuatu

Tumour necrosis factor-alpha (TNF-alpha) is one of the key cytokines that influence the pathology of microbial infections. The genetic susceptibility to severe forms of falciparum malaria is differentially associated with TNF-alpha promoter gene polymorphisms (TNFP alleles). In a previous study, we identified a TNFP-allele characterized by a C to T transition at position -857 (TNFP-D allele) as a marker for susceptibility to cerebral malaria in Myanmar. The frequencies of TNFP alleles on six islands of Vanuatu, Melanesia (South-west Pacific) were estimated to investigate whether malaria selection pressure on this susceptibility marker has influenced its prevalence. Within the archipelago of Vanuatu there is a decreasing cline of parasite incidence from North to South. Of the four alleles of the TNFP gene detected in Vanuatu, the TNFP-D allele frequencies were inversely correlated with the parasite incidence of islands; TNFP-D varied from 0.55 on the island with the lowest parasite incidence to 0.26 on the island with the highest parasite incidence (r = -0.855, P = 0.03). We also observed a significant correlation between the frequencies of alpha-thalassaemia alleles, thought to protect against malaria and parasite incidence in the same populations. These data are consistent with a previously reported correspondence between the frequencies of glucose 6-phosphate dehydrogenase (G6PD) deficiency and parasite incidences on the islands of Vanuatu (Kaneko et al. 1998) and indicate that the degree of malaria endemicity has influenced the allele frequencies of at least three loci that confer both susceptibility (TNFP-D) and protection (alpha-thalassaemias and G6PD deficiency).     

Absence of significant pharmacokinetic and pharmacodynamic interactions between artemether and quinoline antimalarials.

The study was carried out to investigate the pharmacokinetic and pharmacodynamic interactions between artemether (ARTEM) and quinoline antimalarials namely mefloquine (MQ), quinine (QN) and primaquine (PQ) when given concurrently. A randomised comparative, seven way cross-over design was performed in eight healthy male Thais following the administrations of seven drug regimens on seven occasions i.e. a single oral dose of ARTEM (300 mg), or MQ (750 mg), or QN (600 mg), or PQ (45 mg) alone, or the combination of ARTEM (300 mg) with MQ (750 mg), or QN (600 mg), or PQ (45 mg). All clinical and laboratory parameters were normal in all subjects, before, during and after the study. The eight subject experienced no adverse effect after ARTEM, QN, PQ alone regimens, or combination of ARTEM with QN and PQ. After administration of MQ in either occasion, 3 subjects had weakness, nausea, abdominal pain, and diarrhoea; one subject complained of dizziness. All symptoms were mild and occurred during the first day of MQ administration. The fitting of the concentration-time curves of ARTEM, QN and PQ, to a one-compartment model with first order absorption yielded satisfactory results in all subjects. The best fit model for MQ was two-compartment model with first order absorption. The pharmacokinetics of all investigated drug, when given alone or in combination were not significantly different.     

Pharmacokinetics and bioequivalence evaluation of three commercial tablet formulations of mefloquine when given in combination with dihydroartemisinin in patients with acute uncomplicated falciparum malaria

Objective: To assess the pharmacokinetics and relative bioavailability/bioequivalence of three commercial tablet formulations of mefloquine, i.e. Lariam (reference formulation), Mephaquin 100 Lactab and Eloquin-250, when given sequentially after dihydroartemisinin in Thai patients with acute uncomplicated falciparum malaria. Methods: Twenty-nine Thai patients with acute uncomplicated falciparum malaria were randomised to receive an initial dose of 300 mg dihydroartemisinin, followed by 1250 mg mefloquine (at 24 h and 30 h after dihydroartemisinin) given as either Lariam (n=10 cases), Mephaquin (n=9 cases) or Eloquin-250 (n=10 cases). Serial blood samples were obtained up to day 42 after treatment with mefloquine. Mefloquine concentrations were determined in whole blood by means of ultraviolet high-performance liquid chromatography. The pharmacokinetic parameters of mefloquine were estimated using non-compartmental and compartmental analysis. Results: The three combination regimens were well tolerated. Patients in all treatment groups had a rapid initial response. However, nine patients (four and five cases in regimen containing Mephaquin 100 Lactab and Eloquin-250, respectively) had reappearance of parasitaemia during the follow-up period. Mefloquine from the three formulations showed significantly different pharmacokinetic and bioavailability metrics. Significantly lower peak plasma concentrations (C_max) and areas under the plasma concentration–time curve (AUC; AUC_0–48h, AUC_0–7days, and total AUC) were observed with Mephaquin 100 Lactab than with the other two formulations. Mean values for relative bioavailability of the test to standard products were 49.1% (Mephaquin 100 Lactab) and 72.4% (Eloquine-250). Based on the criteria set, the bioavailability of the two test products (Mephaquin 100 Lactab and Eloquine-250) was considered non-equivalent to the reference product with respect to the rate (t_max, C_max) and extent (AUC_0–48h, AUC_0–7days, total AUC) of mefloquine absorption. Received: 15 June 1999 / Accepted in revised form: 7 October 1999     

Plasmodium vivax Isolates from Cambodia and Thailand Show High Genetic Complexity and Distinct Patterns of P. vivax Multidrug Resistance Gene 1 (pvmdr1) Polymorphisms

Plasmodium vivax accounts for an increasing fraction of malaria infections in Thailand and Cambodia. We compared P. vivax genetic complexity and antimalarial resistance patterns in the two countries. Use of a heteroduplex tracking assay targeting the merozoite surface protein 1 gene revealed that vivax infections in both countries are frequently polyclonal (84%), with parasites that are highly diverse (H_E = 0.86) but closely related (G_ST = 0.18). Following a history of different drug policies in Thailand and Cambodia, distinct patterns of antimalarial resistance have emerged: most Cambodian isolates harbor the P. vivax multidrug resistance gene 1 (pvmdr1) 976F mutation associated with chloroquine resistance (89% versus 8%, P < 0.001), whereas Thai isolates more often display increased pvmdr1 copy number (39% versus 4%, P < 0.001). Finally, genotyping of paired isolates from individuals suspected of suffering relapse supports a complex scheme of relapse whereby recurrence of multiple identical variants is sometimes accompanied by the appearance of novel variants.     

Comparative clinical trial of four regimens of dihydroartemisinin-mefloquine in multidrug-resistant falciparum malaria

We conducted a randomized, comparative trial at the Bangkok Hospital for Tropical Diseases during 1996-98 to evaluate the clinical efficacy and tolerability of four combination regimens of dihydroartemisinin-mefloquine. 207 male patients aged 18-25 years, weighing 49.3-55.1 kg were randomized to receive a single oral dose of 300 mg dihydroartemisinin plus one or two doses of mefloquine as follows: regimen I (n = 26): 750 mg mefloquine concurrently, or regimen II (n = 22): 750 mg mefloquine 24 h later, or regimen III (n = 78): 750 and 500 mg mefloquine at 24 and 30 h, or regimen IV (n = 81): 750 and 500 mg mefloquine (at 0 and 24 h). All patients improved clinically within 24 h of initiation of treatment. The initial therapeutic response was rapid and identical in all treatment groups (median PCT vs. FCT: 36 vs. 24, 36 vs. 28, 36 vs. 26, and 34 vs. 26 h, for regimen I, II, III and IV, respectively). All combination regimens generally showed acceptable tolerability profiles. Compliance with follow-up (42 days) was achieved by 86.5% (179 cases). Recrudescent parasitaemia was significantly higher in patients treated with low-dose mefloquine combinations (regimens I, II:8/23, 9/16) than in those who received high-dose mefloquine (regimens III, IV: 2/70, 3/70). No RII or RIII type of response was observed. There were no significant differences in susceptibility to mefloquine between primary and recrudescent isolates. Dose-adjusted whole blood mefloquine concentrations were significantly higher in high-dose mefloquine regimens (III and IV). Patients who vomited within the first hour of mefloquine administration had markedly lower whole blood mefloquine concentrations than those who did not vomit.     

Microsatellite polymorphism in the heme oxygenase-1 gene promoter is associated with susceptibility to cerebral malaria in Myanmar

Cerebral malaria (CM) is a serious complication of Plasmodium falciparum malaria, and its pathogenesis leading to coma remains unknown. Heme oxygenase-1 (HO-1) catalyzes heme breakdown, eventually generating bilirubin, iron and carbon monoxide. The HO-1 gene promoter contains a polymorphic (GT)n repeat which may influence the expression level of HO-1. To explore the correlation between this (GT)n polymorphism and susceptibility to CM, we analyzed the frequencies of the (GT)n alleles in 120 Myanmarese patients with uncomplicated malaria (UM) and 30 patients with CM. The frequency of homozygotes for the short (GT)n alleles (<28 repeats) in CM patients was significantly higher than those in UM patients (P < 0.008, OR = 3.14). Thus, short (GT)n alleles represent a genetic risk factor for CM.     

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).     

Strong association of a tumor necrosis factor-alpha promoter allele with cerebral malaria in Myanmar

To investigate the host genetic factors affecting the clinical course of falciparum malaria, polymorphism of the tumor necrosis factor-alpha (TNF-alpha) promoter region was analyzed in patients with cerebral malaria. Two hundred and forty-three Myanmar patients with falciparum malaria at Mae Sot Malaria clinic and Mae Sot General Hospital located at the border between Thailand and Myanmar, were included in this study. Among the patients (128 from Karen, 115 from Burma), 200 were uncomplicated and 43 had cerebral malaria. The TNF-alpha 5'- flanking region showed biallelic polymorphic sites at -238, -308, -857, -863, -1031, and there were 7 alleles (TNFP-A, B, C, D, M1, M4, M7) found in the patients from Myanmar. We found that the TNFP-D allele was significantly associated with cerebral malaria in the populations from Karen (Pc<0.0001, OR=124.86) and Burma (Pc<0.0001, OR=34.50). TNFP-D showed no significant linkage disequilibrium with any alleles of HLA-B or HLA-DRB1, suggesting that TNFP-D was primarily associated with cerebral malaria in Myanmar.