Am. J. Trop. Med. Hyg., 60(2), 1999, pp Copyright 1999 by The American Society of Tropical Medicine and Hygiene

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1 Am. J. Trop. Med. Hyg., 60(2), 1999, pp Copyright 1999 by The American Society of Tropical Medicine and Hygiene A RANDOMIZED, DOUBLE-BLIND, COMPARATIVE TRIAL OF A NEW ORAL COMBINATION OF ARTEMETHER AND BENFLUMETOL (CGP 56697) WITH MEFLOQUINE IN THE TREATMENT OF ACUTE PLASMODIUM FALCIPARUM MALARIA IN THAILAND S. LOOAREESUWAN, P. WILAIRATANA, W. CHOKEJINDACHAI, K. CHALERMRUT, W. WERNSDORFER, B. GEMPERLI, I. GATHMANN, AND C. ROYCE Department of Clinical Tropical Medicine and Hospital for Tropical Diseases, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; Institut für Spezifische Prophylaxe und Tropenmedizin, University of Vienna, Vienna, Austria; Novartis Pharma AG, Basel, Switzerland Abstract. CGP 56697, a new oral fixed combination of artemether and benflumetol, was tested in a double-blinded, randomized trial in 252 adult patients treated either with CGP (4 4 tablets each containing 20 mg of artemether and 120 mg of benflumetol, given at 0, 8, 24, and 48 hr), or with mefloquine (three tablets of 250 mg at initial diagnosis, followed by two tablets of 250 mg at 8 hr). Baseline data of the two groups were comparable. The 28-day cure rate with CGP was lower than with mefloquine (69.3% versus 82.4%; P 0.002). However, CGP was more effective than mefloquine in parasite clearance time (43 hr versus 66 hr; P 0.001) fever clearance time (32 hr versus 54 hr; P 0.005), and gametocyte clearance time (152 hr versus 331 hr; P 0.001). This study revealed that CGP is effective against multidrug-resistant Plasmodium falciparum malaria in Thailand, but higher doses will probably be needed to improve the cure rate. 238 Each year an estimated 300 million to 500 million cases of malaria occur in the tropics, with between 1.5 million and 2.7 million resultant deaths. 1 It is estimated that this figure will increase to seven million should quinine-resistant forms spread from Southeast Asia. 2 Since 1981, malaria has caused between 20 million and 40 million deaths compared with 2.5 million from acquired immunodeficiency syndrome (AIDS). 1 Soldiers based in malaria-endemic areas have suffered more casualties from malaria than bullets in every campaign of the 20th century, 3 and malaria remains a threat to travelers arriving in or returning from areas endemic for this disease. Of the four pathogenic malarial parasites, Plasmodium falciparum is the most dangerous and the main cause of death. Chloroquine replaced quinine as the major anti-malarial drug in the late 1940s, but within little more than a decade resistant forms had evolved in Thailand and Cambodia. These have now extended to other endemic regions. 4 Similarly, with new drugs to replace chloroquine, such as mefloquine, resistant forms are now emerging in Southeast Asia. 5 Treatment of P. falciparum malaria in Southeast Asia is increasingly difficult and is a particular problem in Thailand. Resistance to all available standard antimalarials is well documented. 5 Quinine plus tetracycline given for seven days is a standard regimen for highly multidrug-resistant P. falciparum strains for admitted patients in Thailand. However, the cure rate with this combination varies between 90% and 98%, even though drug administration was well supervised with treatment given in the hospital. 6,7 Both drugs have a short half-life (6 8 hr), necessitating frequent dosing. In addition, quinine causes predictable side effects such as cinchonism. Both factors make treatment with quinine and tetracycline inconvenient and cause major problems with treatment compliance, especially in the outpatient setting. Mefloquine (15 mg/kg) in adult patients produced a satisfactory response with cure rates of 98% from 1983 to ,9 In 1990, the efficacy of mefloquine at the 15 mg/kg dose level had decreased to 71%. 10 The higher dose of mg/kg of mefloquine produced cure rates of 98% in children and 74% in adults in a recent study High-dose mefloquine (25 mg/kg) plus artesunate is currently used for the treatment of multidrug-resistant falciparum malaria on the eastern and western borders of Thailand. For more than 2,000 years the Chinese have used a wormwood plant (Artemisia annua) extract, artemisinin (qinghaosu), for the treatment of fever. In the 1970s, Chinese scientists identified the structure of the active component against malaria as a sesquiterpene lactone peroxide. More active derivatives such as oil-soluble artemether (methyl ether) and water-soluble artesunate (a hemisuccinate) have been developed and used in monotherapy or in combination with other antimalarials Currently, more than two million people have received anti-malarial treatment with artemisinin, artesunate, or artemether. 21 These drugs are of considerable therapeutic significance because of their activity against both quinine- and chloroquine-resistant falciparum malaria. To date, they are remarkably nontoxic in humans 21,22 have and show fast parasite clearance but monotherapy demonstrates high recrudescence rates of 12 55% depending on dose, duration of treatment, and severity of the disease. 13,20,23,24 In the early 1980s, Chinese scientists at the Beijing Academy of Military Medical Sciences (AMMS) discovered the anti-malarial benflumetol, a dichlorobenzylidine derivative (unpublished data). In contrast to artemether, its onset of activity was slower, it had a longer half-life, and it produced complete parasite clearance (unpublished data). This led to the idea that a highly effective anti-malarial treatment could be achieved by uniting the benefits of both in a combination therapy of artemether plus benflumetol. Initially, investigations at the AMMS (unpublished data) on a similarly formulated combination therapy demonstrated a 28-day cure rate of 97.4% in acute uncomplicated falciparum malaria in China with the four doses given over a 48-hr period. This was confirmed in a Phase II trial in a chloroquine-resistance area in Chinese patients sponsored by Ciba/Novartis (Basel, Switzerland), which gave a 28-day cure rate of 96.1% with the same four doses given over a 48-hr period. 25 We report

2 CGP VERSUS MEFLOQUINE FOR TREATMENT OF P. FALCIPARUM MALARIA 239 TABLE 1 Demographic and baseline data of patients CGP Mefloquine Sex (males) No. (%) 72 (57%) 80 (63%) Age (years) Median Range Weight (kg) Median Mean Range Hematocrit (%) Median Mean Hematocrit 25% Range No. (%) 23 (18%) 18 (14%) G6PD* deficient No. (%) 10 (8%) 8 (6%) Previous malaria infection No. (%) 9 (7%) 8 (6%) Months since previous infection No. (%) 6 (5%) 6 (5%) 1 month 3 (2%) 0 (0%) 1 6 months 0 (0%) 2 (2%) 6 months Hepatomegaly No. (%) 64 (51%) 71 (56%) Splenomegaly No. (%) 29 (23%) 32 (25%) Temperature ( C) Median Range Parasite density (/ l) Median 15,825 18,560 Geometric mean 16,246 17,792 Range 1, ,260 1, ,380 No. (%) 5, (18%) 31 (25%) 5,000 15, (30%) 26 (21%) 15,000 50, (33%) 36 (29%) 50, (19%) 33 (26%) * G6PD glucose-6-phosphate dehydrogenase. here a randomized, double-blind, comparative trial of the combination of artemether and benflumetol (CGP 56697) using the same doses as in China compared with mefloquine (25 mg/kg) in adults in Thailand. PATIENTS AND METHODS Patients with uncomplicated falciparum malaria admitted to the Bangkok Hospital of Tropical Diseases between May 1995 and March 1996 were selected for this study if they were more than 12 years of age, weighed not less than 35 kg, had microscopic confirmation of P. falciparum infection with parasitemias of 1, ,000/ l (or if a mixed infection, to include P. falciparum), and had given written informed consent to participate in the trial. Reasons for exclusion were pregnancy/lactation, inability to tolerate oral administration, signs of severe/complicated malaria, 26 other treatment for malaria in preceding two weeks or use of any investigational drug during the preceding 30 days, hypersensitivity or allergy to artemesinin or mefloquine, a history of epilepsy or neuropsychiatric disease, and severe renal, hepatic, or cardiac impairment. Suitable patients were treated either with CGP or mefloquine in a randomized scheme using a double-dummy technique to blind the trial. This study was approved by the Ethics Committee of Mahidol University. Before the start of treatment, each patient was given a general medical examination and the baseline data were recorded (Table 1). Routine hematology and biochemistry tests were performed, including a glucose-6-phosphate dehydrogenase (G6PD) assay. Pretreatment investigations included a full blood count, serum electrolytes, total and direct bilirubin, alkaline phosphatase, blood urea nitrogen, creatinine, albumin, globulin, and aspartate and alanine aminotransferases, and urine analysis. These tests were repeated on days 1, 3, 7, and 28. To search for adverse effects, thorough daily examinations and repeat electrocardiograms were done on days 1, 3, 7 and 28. Neurologic examinations were done and signs and symptoms were recorded on a standard form daily for the first seven days and weekly thereafter. Adverse effects were defined as signs and symptoms that first occurred or became more severe after treatment started. Parasitologic examination of thick blood films was done every 6 hr after starting treatment during the first 72 hr, then daily for the first seven days and weekly for four weeks. Malaria parasite counts per microliter were obtained by calculation against the red blood cell count for a thin film or against the white blood cell count for a thick film, using Giemsa-stained thick and thin films. Geometric mean parasite counts of the two groups were used as a standard method. Thin films were used to confirm the presence of Plasmodium species other than P. falciparum. Whenever gametocytes were found they were counted per 1,000 leukocytes. Blood films were considered negative if no parasites were seen in 200 oil-immersion fields in a thick blood film. Dosage and administration. CGP was given in the following regimen: each tablet contained 20 mg of artemether and 120 mg of benflumetol; each patient received four doses of four tablets each, the first given at 0 hr, then

3 240 LOOAREESUWAN AND OTHERS at 8 hr, 24 hr, and 48 hr. Thus, each dose comprised 80 mg of artemether and 480 mg of benflumetol, with a total of 320 mg of artemether and 1,920 mg of benflumetol given to each patient over the 48-hr treatment period. Mefloquine was given in a standard treatment course: each tablet contained 250 mg of base, the treatment course totalling five tablets with three (750 mg) given at 0 hr and two (500 mg) given 8 hr later (total course 1,250 mg). Trial medications for each patient (active and placebo) were individually boxed and coded via a computer-generated randomization. All personnel involved were blind to drug trial codes until trial completion. Patients who had vomited the trial medication within 1 hr were redosed. Outcome measures. The response to treatment was characterized by 1) the 28-day cure rate, 2) parasite clearance time (PCT), 3) fever clearance time (FCT), and 4) gametocyte clearance time. Parasite clearance time was the time from the start of treatment until the first time blood films were negative and remained negative for the next 48 hr. Fever clearance time was the time from the start of treatment until the oral temperature decreased to 37.5 C and remained below this temperature during the next 48 hr. Gametocyte clearance time was the time from the start of treatment in patients who were gametocyte positive in blood smears at the beginning of the trial until the first time blood films were negative and remained negative throughout the 28-day follow-up. The 28-day cure rate was defined as the absence of a recrudescence during the 28-day follow-up, and treatment failures were categorized into RI, RII, or RIII failures. 27 All patients were admitted to hospital and remained there for 28 days to exclude reinfection. Treatment failures. Patients who recrudesced with P. falciparum were treated by the standard regimen used in our hospital, i.e., artesunate, 600 mg followed by mefloquine, 1,250 mg (both total doses). 14 Patients who relapsed with asexual forms of P. vivax were treated with a single dose of 450 mg (base) of chloroquine. They were not withdrawn from the trial and were included in the final evaluation since P. falciparum in Thailand is uniformly resistant to chloroquine. 5 Primaquine, 15 mg base, was given once a day for 14 days after the patient completed 28 day follow-up (end of the clinical trial). Statistical analysis. The method of Casagrande 28 was used to calculate the minimum sample size needed for comparison of the 28-day cure rates, giving 113 for each group, assuming a 95% cure rate for CGP and 80% for mefloquine, using 5%, power 90%, and allowing for a drop-out rate of 10% during the trial. The 95% confidence intervals (CIs) for each cure rate were calculated using Pearson-Clopper limits and the proportions were compared using Fisher s exact test. The median and 95% CIs for the median were calculated for PCT, FCT, and gametocyte clearance using the Kaplan-Meier method, and the treatment effect was tested using the Wilcoxon test. Additional adjusted analyses of PCT and FCT in relation to baseline data were made using Cox s proportional hazard regression. The percentage parasite reduction compared with baseline was analyzed using the Wilcoxon ranksum test. RESULTS Of the 252 enrolled patients, 126 were randomly assigned to the CGP group and 126 to the mefloquine group. Baseline clinical data of the two groups were comparable and are shown in Table 1. Overall, 233 were evaluable for the 28-day cure rate since 19 (12 patients in GCP treated group and seven in the mefloquine-treated group) were lost to follow-up due to social reasons not related to drug treatment. All 19 patients had no parasitemias and were well before they left hospital. Efficacy results of the treatment groups are shown in Table 2. With a 28-day cure rate of 82.4%, mefloquine produced a better result than CGP 56697, which showed a cure rate of 69.3% (P 0.022). When the cure rates of the patients were examined in relation to their baseline characteristics (parasite density, age, temperature, weight, G6PD, sex, history of previous infection) and treatment, the only relationship found in addition to the treatment effect was that the 28-day cure rate was better 1) when initial parasite levels were low, i.e., less than 5,000/ l (P 0.001) and/or 2) in older patients, i.e., more than 30 years of age (P 0.02). Parasite clearance time and fever clearance time of the mefloquine- and CGP treated groups were significantly shorter in the CGP group (66 hr versus 43 hr and 54 versus 32 hr, respectively; P 0.01). There was no RII or RIII failures in CGP treated group. Four patients receiving mefloquine experienced RIII failures and needed rescue medication within the first two days. An additional five patients receiving mefloquine did not clear parasites within the first week (RII failures) and needed rescue medication. 14 If parasite clearance was not achieved by the time of the final blood film, or if other anti-malarial drugs were given due to resistance to mefloquine, then the data of these patients were censured (n 11): 10 (8%) from the mefloquinetreated group and one (1%) from the CGP treated group. The median PCT for CGP (43 hr) was significantly shorter than for mefloquine (66 hr; P 0.001, by the Wilcoxon test, Figure 1). An exploratory adjusted analysis (Cox s proportional hazard regression) showed that only the baseline parasite density and temperature of the patients affected the PCT (P 0.01). The markedly superior PCT values for CGP versus mefloquine remained even when allowing for these baseline adjustments. The estimated parasite reduction at 24 hr compared with baseline was 98.6% for CGP (C) versus 76.1% for mefloquine (P 0.001, by the Wilcoxon rank-sum test). One hundred forty-two patients were evaluable for the FCT; the other 110 patients had baseline temperatures 37.5 C. The FCT was censured at the last temperature measurement if it was not maintained at 37.5 C for at least 48 hr (n 6). The overall data are summarized in Table 2 and show the superiority of CGP (32 hr) compared with mefloquine (54 hr) in the FCT (P 0.003, by the Wilcoxon test). Cox s proportional hazard regression showed that the only baseline characteristic affecting FCT was parasite density (P 0.003). The treatment superiority remained significantly in favor of CGP (P 0.015, by Cox s regression) even when allowing for this factor.

4 CGP VERSUS MEFLOQUINE FOR TREATMENT OF P. FALCIPARUM MALARIA 241 TABLE 2 Therapeutic responses* CGP Mefloquine No. of patients with 28-day follow up No. of patients cured at 28 days % of patients cured (95% CI) 69.3% (60.0, 77.6)% 82.4% (74.3, 88.7)% No. (%) with RI response 35 (30.7%) 12 (10.1%) No. (%) with RII response 5 (4.2%) No. (%) with RIII response 4 (3.4%) Number of patients evaluated for PCT PCT (hr) Median (95% CI) 43 (42, 45) 66 (61, 70) Range Number of patients evaluated for FCT FCT (hr) Median (95% CI) 32 (23, 37) 54 (36, 66) Range Number of patients with gametocytes Gametocyte clearance time (hr) Median (95% CI) 152 (89, 185) 331 (281, 492) Range *CI confidence interval; PCT parasite clearance time; FCT fever clearance time. P 0.022, by Fisher s exact test. P 0.001, by Kaplan-Meier method, Wilcoxon test. P 0.003, by Kaplan-Meier method, Wilcoxon test. P 0.001, by Kaplan-Meier method, Wilcoxon test. FIGURE 1. Time to parasite clearance plot against time after initiation of treatment (Kaplan-Meier estimate of survival function). Mean time to parasite clearance 43 hr by CGP versus 66 hr by mefloquine (n 166); P

5 242 LOOAREESUWAN AND OTHERS Gametocyte clearance. Overall, 62 (49.2%) of patients in the group receiving CGP and 74 (58.7%) of the patients receiving mefloquine had gametocytes in their blood at the beginning of the trial (within first 72 hr). Table 2 summarizes the gametocyte clearance data for these 136 patients. All patients receiving CGP who were negative for gametocytes at baseline remained negative throughout the trial except for one patient who had a single slide positive for gametocytes on day 8. Ten patients receiving mefloquine who were negative at baseline developed gametocytes during the trial. The time to gametocyte clearance was calculated as the time from the first dose until gametocytes had disappeared for at least 48 hr. The data were censured for 27 patients not achieving gametocyte clearance (including 11 patients who were lost to follow-up and five patients with treatment failures necessitating alternative anti-malarial medication before gametocyte clearance). The time to gametocyte clearance of the 136 evaluable patients was 152 hr for those receiving CGP and 331 hr for receiving mefloquine (P 0.001, by the Wilcoxon test). A total of 45.2% of the patients receiving CGP reached gametocyte clearance within 96 hr compared with only 17.6% for those receiving mefloquine. Adverse effects. There were no deaths during the trial. Most adverse events were of mild to moderate severity. The adverse event was recorded as severe in two patients: one receiving CGP had severe fatigue on the day of presentation, and one receiving mefloquine had pneumonia (days 6 8). Both events were obviously not drug-associated. More than 70% of the patients had typical malaria symptoms of asthenia, rigors, anorexia, nausea, dizziness, and headache at baseline, and more than 50% presented with vomiting prior to drug administration. All of these symptoms resolved with treatment in both groups. Vomiting within 1 hr of dosing, which required redosing, occurred in nine patients (one patient receiving CGP and eight patients receiving mefloquine). One female patient receiving mefloquine had a serious adverse event with rapid deterioration, but it was shown that the condition was AIDS-related. None of the patients developed any new major cardiovascular symptoms or signs (electrocardiogram [ECG]) after baseline. At baseline, 10.3% of the patients in the CGP group and 18.3% of the patients in the mefloquine group had nonspecific T-wave abnormalities. After treatment, a few patients had nonspecific T waves and ST elevation occurred during the 28-day follow-up (4.8% in the CGP group and 6.3% in the mefloquine group), but these were assessed as not clinically important. The QTc was unchanged in both treatment groups. Several patients had abnormal hematology and biochemistry values at baseline, but all of these improved 1 2 weeks after treatment. There were no physical complaints or abnormal laboratory findings, other than those normally associated with malarial infections, and no abnormalities attributable to either trial treatment. There was no evidence of bone marrow suppression or hepatic or renal toxicity with the use of CGP The ECG monitoring revealed no untoward effects from either mefloquine or CGP therapy. DISCUSSION The results of this double-blind, randomized trial in adult patients in Thailand confirm the rapid and superior parasite reduction and clearance by CGP 56697, with a median PCT at 43 hr, a 98.6% reduction in parasite density within 24 hr, and a median FCT of 32 hr, compared with mefloquine. Of considerable importance were the observations on gametocyte clearance with CGP 56697, which showed a more rapid reduction in gametocyte numbers compared with mefloquine. Half of the CGP treated patients had achieved gametocyte clearance within 96 hr, with the median time to complete gametocyte clearance (152 hr) being less than half that with mefloquine (331 hr). These results confirm the recent report indicating that artemesinin-containing regimens may have important potential for reducing transmission. 29 The recrudescence rates after treatment with artemisinin derivatives alone varied greatly from 12% to 55% depending on dose, duration of treatment, and severity of disease, with the more severe cases at greater risk of having a recrudescence. 13,19,20,23,24 In this study, a combination of artemether with benflumetol (CGP 56697) showed a 28-day cure rate of 69.3%, although recent studies in China with CGP (n 155) showed 28-day cure rates 95%, with very rapid parasite reduction and clearance using the same dosage (AMMS, unpublished data). The lower cure rate obtained in our study might result from greater severity of infection and the highly resistant parasites in this area. Most of our patients were nonimmune and more than 95% were experiencing their first malaria attack. Although the overall cure rate with CGP was lower than that with mefloquine, no high-grade treatment failures were observed, whereas with mefloquine, four patients had potently life-threatening RIII failures. This indicates that some highly mefloquine-resistant P. falciparum are prevalent on the borders of Thailand. The poorer 28-day cure rate of CGP compared with mefloquine might be explained by the parasites from Thailand being more resistant than the parasites from China, or because the duration of treatment was too short for the parasites from Thailand. The required duration of artemisinin derivatives used as monotherapy for the treatment of falciparum malaria in Thailand is at least 5 7 days. Furthermore, the follow-up in this study was only 28 days, and more patients might recrudesce in the mefloquine-treated group after day 28. However, this study indicates that an increase in the dosage of CGP above that found to be effective in China may be necessary in areas of multidrug resistance such as Thailand. Trials with higher dosage regimens are in progress. While the dosage of CGP tested in this trial may have been suboptimal, its excellent tolerability, especially with regard to vomiting, safety profile, and rapid action in clearing parasites from the blood indicate a very promising new combination treatment for malaria. Overall, the results of this study indicate that the combination of artemether and benflumetol is effective and well tolerated but requires further study to define the optimum dosage for use in Thailand.

6 CGP VERSUS MEFLOQUINE FOR TREATMENT OF P. FALCIPARUM MALARIA 243 Financial support: This trial was supported by Novartis Pharma as part of the international development program for CGP Authors addresses: S. Looareesuwan, P. Wilairatana, W. Chokejindachai, and K. Chalermrut, Department of Clinical Tropical Medicine and Hospital for Tropical Diseases, Faculty of Tropical Medicine, Mahidol University, 420/6 Rajvithi Road, Bangkok, Thailand. W. Wernsdorfer, Institut für Spezifische Prophylaxe und Tropenmedizin, University of Vienna, A-1095 Vienna, Austria. B. Gemperli, I. Gathmann, and C. Royce, Novartis Pharma AG, CH Basel, Switzerland. Reprint requests: S. Looareesuwan, Faculty of Tropical Medicine, Mahidol University, 420/6 Rajvithi Road, Bangkok 10400, Thailand. REFERENCES 1. Day M, Malaria falls to herbal remedy. New Scientist 151: Hoffmann SL, Artemether in severe malaria still too many deaths (editorial). 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