A Drug Repurposing Approach Reveals Targetable Epigenetic Pathways in Plasmodium vivax Hypnozoites.

Radical cure of Plasmodium vivax malaria must include elimination of quiescent 'hypnozoite' forms in the liver; however, the only FDA-approved treatments are contraindicated in many vulnerable populations. To identify new drugs and drug targets for hypnozoites, we screened the Repurposing, Focused Rescue, and Accelerated Medchem (ReFRAME) library and a collection of epigenetic inhibitors against P. vivax liver stages. From both libraries, we identified inhibitors targeting epigenetics pathways as selectively active against P. vivax and P. cynomolgi hypnozoites. These include DNA methyltransferase (DNMT) inhibitors as well as several inhibitors targeting histone post-translational modifications. Immunofluorescence staining of Plasmodium liver forms showed strong nuclear 5-methylcystosine signal, indicating liver stage parasite DNA is methylated. Using bisulfite sequencing, we mapped genomic DNA methylation in sporozoites, revealing DNA methylation signals in most coding genes. We also demonstrated that methylation level in proximal promoter regions as well as in the first exon of the genes may affect, at least partially, gene expression in P. vivax. The importance of selective inhibitors targeting epigenetic features on hypnozoites was validated using MMV019721, an acetyl-CoA synthetase inhibitor that affects histone acetylation and was previously reported as active against P. falciparum blood stages. In summary, our data indicate that several epigenetic mechanisms are likely modulating hypnozoite formation or persistence and provide an avenue for the discovery and development of improved radical cure antimalarials.

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).

Association of adhesion molecule PECAM-1/CD31 polymorphism with susceptibility to cerebral malaria in Thais.

Adhesion molecules on endothelial cells are known to be important ligands for malaria infected red blood cells (PRBC) [Mol Biochem Parasitol, 76, (1996) 1], and may be involved in the pathogenic process of cerebral malaria (CM) which is the most serious complication of falciparum malaria, through enhancing micro embolism or sequestration in the capillaries of the brain. PECAM-1/CD31 is one of these candidate ligands and is coded by a polymorphic gene. Two hundred and ten Thai malaria patients (43 cerebral, 89 severe and 78 uncomplicated) were analyzed for their genetic polymorphism of CD31 to examine the clinical relationship between the disease and specific genotypes. Four alleles were defined 125 valine (V)-563 asparagine (N); 125V-563 serine (S); 125 leucine (L)-563N; and 125L-563S. We found that the frequency of the 125 V/V 563 N/N genotype was significantly high in CM patients as compared with severe cases without CM (P<0.01, OR=2.92), suggesting that this genotype is one of the risk factors for CM.

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.

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.

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.

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.

Pharmacokinetic and pharmacodynamic interactions of mefloquine and dihydroartemisinin.

Pharmacokinetic and pharmacodynamic interactions between dihydroartemisinin and mefloquine were investigated in 10 healthy Thai males. The study was of a three-way crossover design. Subjects were randomized to receive three drug regimens on three separate occasions as follows: regimen I: a single oral dose of 300 mg dihydroartemisinin; regimen II: a single oral dose of 750 mg mefloquine; regimen III: a single oral dose of 300 mg dihydroartemisinin, given concurrently with a single oral dose of 750 mg mefloquine. All regimens were well tolerated. Oral dihydroartemisinin was rapidly absorbed and disappeared from systemic circulation within 8-10 h. Mefloquine absorption and disposition were relatively slow processes. Pharmacokinetics of dihydroartemisinin and mefloquine when given concurrently were similar, except for the absorption rate of mefloquine which was faster in the presence of dihydroartemisinin. Pharmacodynamically, the combination of dihydroartemisinin and mefloquine resulted in a synergistic effect on ex vivo blood schizontocidal activity. Maximum activity (Amax) and area under effect-time curve (AUEC) of dihydroartemisinin and mefloquine were increased approximately two- and 20-fold (Amax), and four- and twofold, respectively, compared with each individual drug alone. AUEC of mefloquine during the first 24 h (AUEC 0-24 h) was increased approximately 50-fold in the presence of dihydroartemisinin.

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.

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).

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.

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.

Alternative method for determination of pyrimethamine in plasma by high-performance liquid chromatography.

A rapid, selective, sensitive and reproducible reversed-phase high-performance liquid chromatography (HPLC) procedure for the quantitative determination of pyrimethamine (PYR) in plasma is described. The procedure involved the two-step extraction of PYR and the internal standard, quinine (QN) with acetonitrile and dichloromethane at basic pH. Chromatographic separation consisted of the mobile phase (methanol-water containing 0.005 M octanesulfonic acid, 50:50, v/v) running through the column (Techopak-10 C18) at a flow-rate of 1.6 ml/min. Detection was at UV wavelength of 240 nm. The mean recoveries of PYR and QN at a concentration range of 50 and 500 ng/ml were 98.9 and 89%, and 94.7 and 96% for PYR and QN. The within-day coefficients of variation were 2.1-5.1% for PYR and 5.9% for QN. The day-to-day coefficients of variation were 2.1-4.1% for PYR and 5% for QN. The minimum detectable concentrations for PYR and QN in plasma were 3 and 10 ng/ml. The method was found to be suitable for use in clinical pharmacokinetic study.

In vitro blood schizonticidal activity of sera containing mefloquine-quinine against Plasmodium falciparum.

Serum samples collected at intervals from healthy volunteers, after the administration of 3 drug regimens (quinine (QN) 600 mg, mefloquine (MQ) 750 mg, and MQ 750 mg plus QN 600 mg) were investigated for their blood schizonticidal activities against K1 strain Plasmodium falciparum in vitro. Superiority of activity was shown in the sera collected after the combination regimen. In the diluted sera of the QN regimen, a complete inhibitory effect was observed for only 24 hours, whereas the effect was sustained for 72 hours in the sera collected after MQ in either regimen (MQ alone or MQ/QN). The pattern of minimum inhibitory concentrations (MICs) of QNEq of the sera from QN alone was constant throughout a 24-hour period, with significantly higher concentrations than that from the combination regimen (118-150 vs 21.25-73.5 micrograms/l). In sera collected after the combination regimen, however, the MIC gradually decreased from 0.5 until 2.5 and 4 h, and thereafter gradually returned to the same levels again during a period of 6-24 hours. The MICs of MQEq when given as MQ alone or in combination appeared constant, with a significantly higher value in the former regimen (24.4-26.8 vs 17-19.2 micrograms/l).

Pharmacokinetics and ex vivo anti-malarial activity of sera following a single oral dose of dihydroartemisinin in healthy Thai males.

The pharmacokinetics of dihydroartemisinin (DHA) was studied in eight healthy male Thai subjects after a single oral dose of 300 mg. Absorption of oral DHA was rapid, Cmax of 679 (307-1000) ng/ml was observed at 1.5 (1-2.5) hours after dosing [median (range)]. Plasma concentrations declined monoexponentially and at 12 hours after administration, the levels were below the detection limit (3 ng/ml). A large variation in the AUC (approximately) 50% was observed. The median (range) AUC was 2010 (636-4079) ng h/ml. The lag time and absorption half-life (t1/2a) were 0.169 (0.111-0.277) hours and 0.709 (0.367-1.118) hours respectively. t1/2z was 1.25 (0.79-1.89) hours Vz/f and CL/f were 5.9 (3.5-8.2) l/kg and 45.3 (28.6-122.8) ml/min/kg, respectively. The pattern of its ex vivo serum activity coincided with the plasma concentrations of DHA.