Sant Muangnoicharoen

Identification of a 1,2,4,5-tetraoxane antimalarial drug-development candidate (RKA 182) with superior properties to the semisynthetic artemisinins.

Artemisinin (1) is an extract of the Chinese wormwood Artemisia annua and has been used since ancient times to treat malaria. Today, semisynthetic derivatives artesunate (2) and artemether (3) are used clinically in drug combinations (ACT; artemisinin-based combination therapy). However, first-generation analogues (e.g. 2 and 3) have a limited availability, high cost, and poor oral bioavailability (Scheme 1a). In addition to these drawbacks there have been recent reports of high failure rates associated with ACTs suggesting the possibility of clinical artemisinin resistance along the Thai–Cambodian border. In the light of these observations there is an urgent need to develop alternative endoperoxide-based therapies. The crucial structural functionality within artemisinin and synthetic 1,2,4-trioxanes is the endoperoxide bridge. Recently a series of molecules based on an ozonide structure were developed from which the candidate OZ277 was shown to have impressive antimalarial activity profiles in vitro and in rodent models of malaria. However, the recent

Comparison of the reactivity of antimalarial 1,2,4,5-tetraoxanes with 1,2,4-trioxolanes in the presence of ferrous iron salts, heme, and ferrous iron salts/phosphatidylcholine.

Dispiro-1,2,4,5-tetraoxanes and 1,2,4-trioxolanes represent attractive classes of synthetic antimalarial peroxides due to their structural simplicity, good stability, and impressive antimalarial activity. We investigated the reactivity of a series of potent amide functionalized tetraoxanes with Fe(II)gluconate, FeSO(4), FeSO(4)/TEMPO, FeSO(4)/phosphatidylcholine, and heme to gain knowledge of their potential mechanism of bioactivation and to compare the results with the corresponding 1,2,4-trioxolanes. Spin-trapping experiments demonstrate that Fe(II)-mediated peroxide activation of tetraoxanes produces primary and secondary C-radical intermediates. Reaction of tetraoxanes and trioxolanes with phosphatidylcholine, a predominant unsaturated lipid present in the parasite digestive vacuole membrane, under Fenton reaction conditions showed that both endoperoxides share a common reactivity in terms of phospholipid oxidation that differs with that of artemisinin. Significantly, when tetraoxanes undergo bioactivation in the presence of heme, only the secondary C-centered radical is observed, which smoothly produces regioisomeric drug derived-heme adducts. The ability of these tetraoxanes to alkylate the porphyrin ring was also confirmed with Fe(II)TPP and Mn(II)TPP, and docking studies were performed to rationalize the regioselectivity observed in the alkylation process. The efficient process of heme alkylation and extensive lipid peroxidation observed here may play a role in the mechanism of action of these two important classes of synthetic endoperoxide antimalarial.

Role of Known Molecular Markers of Resistance in the Antimalarial Potency of Piperaquine and Dihydroartemisinin In Vitro

ABSTRACT Using a range of laboratory-adapted and genetically modified Plasmodium falciparum parasite isolates, we investigated the interaction between dihydroartemisinin and piperaquine (PIP), the individual components of an artemisinin combination therapy currently under development, in addition to the role of known drug resistance genes in parasite susceptibility in vitro. All but one parasite line investigated displayed an interaction of dihydroartemisinin and PIP that was antagonistic, although the degree of antagonism was isolate dependent. In terms of resistance markers, the pfcrt haplotypes CVIET and SVMNT were positively associated with reduced sensitivity to PIP, with parasites carrying the South American CQR (SVMNT) allele being generally less sensitive than CVIET parasites. Parasites carrying the CQS (CVMNK) allele displayed a further increase in PIP sensitivity compared with CVIET and SVMNT parasites. Our data indicate that PIP sensitivity was not affected by pfmdr1 sequence status, despite positive correlations between the structurally related compound amodiaquine and pfmdr1 mutations in other studies. In contrast, neither the pfcrt nor pfmdr1 sequence status had any significant impact on susceptibility to dihydroartemisinin.

Indicators of fatal outcome in severe Plasmodium falciparum malaria: a study in a tertiary–care hospital in Thailand

Objective: To illustrate the clinical features and investigate the indicators associated with a fatal outcome in adult patients with severe Plasmodium falciparum malaria admitted to the Hospital for Tropical Diseases, Bangkok, Thailand. Methods: We studied 202 adult malaria patients admitted to the Intensive Care Unit. A total of 43 clinical variables were identified by univariate and logistic regression analyses, to eliminate confounding factors. Results: Regarding the statistical methods, only 6 variables-jaundice, cerebral malaria, metabolic acidosis, body mass index, initial respiratory rate, and white blood cell count-were significant indicators of death, with adjusted odds ratios (95% CI) of 15.2 (2.1-32.3), 4.3 (2.3-12.6), 3.3 (2.3-5.7), 2.4 (1.9-3.5), 2.2 (1.5-2.6), and 1.7 (1.2-3.1), respectively. Conclusions: Our study found that jaundice, cerebral malaria, metabolic acidosis, body mass index, initial respiratory rate and white blood cell count were indicators of fatal outcome in severe Plasmodium falciparum malaria. Further studies on the fatal indicators in severe malaria need to be compared with data from different geographical areas, to construct practical measures to address potentially fatal indicators in different settings.

Plasmodium falciparum strains harboring dihydrofolate reductase with the I164L mutation are absent in Malawi and Zambia even under antifolate drug pressure.

ABSTRACT The Plasmodium falciparum dihydrofolate reductase (PfDHFR) enzyme is the target of pyrimethamine, a component of the antimalarial pyrimethamine-sulfadoxine. Resistance to this drug is associated primarily with mutations in the Pfdhfr gene. The I164L mutant allele is of particular interest, because strains possessing this mutation are highly resistant to pyrimethamine and to chlorproguanil, a component of chlorproguanil-dapsone. A recent study from Malawi reported this mutation at a prevalence of 4.7% in parasites from human immunodeficiency virus-positive pregnant women by using a real-time PCR method. These observations have huge implications for the use of pyrimethamine-sulfadoxine, chlorproguanil-dapsone, and future antifolate-artemisinin combinations in Africa. It was imperative that this finding be rigorously tested. We identified a number of critical limitations in the original genotyping strategy. Using a refined and validated real-time PCR strategy, we report here that this mutation was absent in 158 isolates from Malawi and 42 isolates from Zambia collected between 2003 and 2005.

Oral Activated Charcoal Prevents Experimental Cerebral Malaria in Mice and in a Randomized Controlled Clinical Trial in Man Did Not Interfere with the Pharmacokinetics of Parenteral Artesunate

Background Safe, cheap and effective adjunct therapies preventing the development of, or reducing the mortality from, severe malaria could have considerable and rapid public health impact. Oral activated charcoal (oAC) is a safe and well tolerated treatment for acute poisoning, more recently shown to have significant immunomodulatory effects in man. In preparation for possible efficacy trials in human malaria, we sought to determine whether oAC would i) reduce mortality due to experimental cerebral malaria (ECM) in mice, ii) modulate immune and inflammatory responses associated with ECM, and iii) affect the pharmacokinetics of parenteral artesunate in human volunteers. Methods/Principal Findings We found that oAC provided significant protection against P. berghei ANKA-induced ECM, increasing overall survival time compared to untreated mice (p<0.0001; hazard ratio 16.4; 95% CI 6.73 to 40.1). Protection from ECM by oAC was associated with reduced numbers of splenic TNF+ CD4+ T cells and multifunctional IFNγ+TNF+ CD4+ and CD8+ T cells. Furthermore, we identified a whole blood gene expression signature (68 genes) associated with protection from ECM. To evaluate whether oAC might affect current best available anti-malarial treatment, we conducted a randomized controlled open label trial in 52 human volunteers (ISRCTN NR. 64793756), administering artesunate (AS) in the presence or absence of oAC. We demonstrated that co-administration of oAC was safe and well-tolerated. In the 26 subjects further analyzed, we found no interference with the pharmacokinetics of parenteral AS or its pharmacologically active metabolite dihydroartemisinin. Conclusions/Significance oAC protects against ECM in mice, and does not interfere with the pharmacokinetics of parenteral artesunate. If future studies succeed in establishing the efficacy of oAC in human malaria, then the characteristics of being inexpensive, well-tolerated at high doses and requiring no sophisticated storage would make oAC a relevant candidate for adjunct therapy to reduce mortality from severe malaria, or for immediate treatment of suspected severe malaria in a rural setting. Trial Registration Controlled-Trials.com ISRCTN64793756

Chronic hepatitis B prognostic markers other than pre-treatment viral load predicted composite treatment outcome

INTRODUCTION Chronic hepatitis B (CHB) is a globally common infectious disease. Its clinical course is complicated. In Southeast Asia, nucleos(t)ide analogues (NA) are commonly used drugs for CHB treatment. Composite treatment outcome has often been used in CHB clinical practice, but rarely predicted epidemiologically. This study aimed to compare the composite treatment outcome between CHB patients with low and high treatment-naïve viral load, and to identify its predictors METHODOLOGY This retrospective cohort study followed up 95 CHB patients on NA treatment for a year. Composite treatment outcome was defined as undetectable HBV DNA level, ALT normalization and, HBeAg clearance in the case of HBeAg-positive patients. Multinomial logistic regression analysis was applied to analyze the significant treatment response predictors. RESULTS Complete composite treatment outcome was achieved by 52% of CHB patients with an initial viral load < 6.5 log 10 copies /ml, but 31% of those had an initial viral load ≥ log 6.5 log 10 copies /ml. Outcome was predicted by HBeAg negativity (adjusted relative risk ratio, aRRR = 11.1, 95 % confidence interval, CI 3-41.3) and ALT normalization within the sixth month of therapy (aRRR = 6.7, CI 1.8-24.9). An elevation of ALT to more than 1.5 times the normal value (40 IU/ml) can lead to an incomplete response on NA therapy (aRRR = 6.2, CI 1.5-26.6.) CONCLUSION Routine clinical markers other than pre-treatment viral load predicted composite CHB outcome on NA Therapy.

Atypical lymphocytes in malaria mimicking dengue infection in Thailand

correspondence: Polrat Wilairatana Department of clinical Tropical Medicine, Faculty of Tropical Medicine, Mahidol University, Rajvithi Road, Rajthevi, Bangkok 10400, Thailand Tel +662 354 9159 Fax +662 354 9158 email tmpwl@mahidol.ac.th Abstract: Patients with uncomplicated falciparum or vivax malaria usually present with acute febrile illness and thrombocytopenia similar to dengue infection. We retrospectively studied atypical lymphocytes (AL) and atypical lymphocytosis (ALO, defined as AL . 5% of total white blood cells) in 1310 uncomplicated malaria patients. In 718 falciparum malaria patients, AL and ALO on day 0 were found in 53.2% and 5.7% of the patients, respectively, with median AL on admission of 1% (range 0%–10%), whereas in 592 vivax malaria patients, AL and ALO on day 0 were found in 55.4% and 9.5% of the patients, respectively, with median AL on admission of 1% (range 0%–14%). After antimalarial treatment, AL and ALO declined in both falciparum and vivax malaria. However, AL and ALO remained in falciparum malaria on days 7, 14, and 21, whereas AL and ALO remained in vivax malaria on days 7, 14, 21, and 28. In both falciparum and vivax malaria patients, there was a positive correlation between AL and total lymphocytes, but a negative correlation between AL and highest fever on admission, white blood cells, and neutrophils, eosinophils, and platelets (P , 0.05). In conclusion, AL or ALO may be found in uncomplicated falciparum and vivax malaria mimicking dengue infection. In tropical countries where both dengue and malaria are endemic, presence of AL or ALO in any acute febrile patients with thrombocytopenia (similar to the findings in dengue) malaria could not be excluded. Particularly if the patients have risk of malaria infection, confirmative microscopic examination for malaria should be carried out.