Clinical Efficacy of High Dose Monotherapy of Oral Dihydroartemisinin in Uncomplicated Falciparum Malaria in Viet Nam

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1 Jpn. J. Infect. Dis., 60, , 2007 Original Article Clinical Efficacy of High Dose Monotherapy of Oral Dihydroartemisinin in Uncomplicated Falciparum Malaria in Viet Nam Le Thi Diem Thuy 1,2, Kesara Na-Bangchang 1, Le Ngoc Hung 2 *, Mieu Tieu Chong 3, Nguyen Van Thang 4, Nguyen Van Binh 5 and Phan Thi Danh 2 1 Faculty of Allied Health Sciences, Thammasat University, Pathumthani, Thailand; 2 Cho Ray Hospital, Ho Chi Minh City; 3 Bac Binh District Hospital; 4 Mepu Regional Health Post, Binh Thuan Province; and 5 11th Rubber Plantation Health Post, Binh Phuoc Province, Viet Nam (Received November 7, Accepted March 1, 2007) SUMMARY: The clinical efficacy of the monotherapy involving the administration of a high dose of dihydroartemisinin (DHA 900 mg) for 5 days was compared with that of the combination regimen (DHA 600 mg + mefloquine [MQ] 750 mg) in an open randomized study in 90 patients with uncomplicated falciparum malaria in the southern part of Viet Nam. Patients were randomly treated with the DHA-5 day monotherapy regimen (300, 300, 100, 100, and 100 mg given at 0, 24, 48, 72, and 96 h) or the DHA-MQ combination regimen (300 mg DHA at 0 h, then 300 mg DHA plus 750 mg MQ at 24 h). The end points for comparison were the parasite and fever clearance times (PCT and FCT) and recrudescence rates (by day 28 for DHA-5 days and day 42 for DHA- MQ). Eighty-nine patients completed the trial per protocol, including 45 cases receiving DHA-5 day and 44 receiving DHA-MQ. There was no difference in clinical manifestations, parasitemia density or other laboratory tests between the two patient groups. The PCTs were 35.3 ± 17.4 h (mean ± SD; range, 12-96) and 37.8 ± 19.2 h (range, 12-96), respectively for the DHA-5 day and DHA-MQ regimens (P > 0.05). Twelve patients receiving the DHA-5 day regimen relapsed with falciparum malaria by day 28 (26.7%) and 5 patients receiving the DHA- MQ regimen relapsed by day 42 (11.4%) (P = 0.07). Survival analysis showed that the DHA-5 day regimen had a radical cure rate significantly lower than that of the DHA-MQ regimen (P = 0.003). The high dose of DHA in the monotherapy regimen did not increase the efficacy of the treatment of patients with uncomplicated Plasmodium falciparum malaria. The DHA combination regimens are suggested to be the better regimens for DHA. INTRODUCTION Dihydroartemisinin (DHA) is a semi-synthetic derivative of artemisinin and has been used in the clinical treatment of patients with falciparum malaria in many tropical countries, especially in the Asian region (1). DHA is considered to be a powerfully acting antimalarial drug against Plasmodium falciparum parasites (2). The cost of production of DHA is lower than that of other artemisinin derivatives (3). Thus, this drug seems to have a good potential for use in developing countries. In clinical treatment, the artemisinin derivatives have been used in two ways, in monotherapy or in combination regimens (4). In monotherapy regimens, DHA was used in treatment courses of 3 to 5 days. The radical cure rates for monotherapy with artesunate, artemether and DHA were reported to be as high as 90% in some clinical trials (5-7), but were as low as 80% or less in other reports (8-10). The artemisinin derivatives, including DHA, have a short half-life (e.g., DHA, min) (4). Therefore, these drugs disappear quickly from the body, within a few hours after administration. In addition, declining concentrations of DHA in plasma have been reported during 5-day oral treatment with artesunate for falciparum malaria (11). These findings suggest that the antimalarial activity of artemisinin derivatives is short-lived. However, a continuous reduction in the *Corresponding author: Mailing address: Department of Clinical Biochemistry, Cho Ray Hospital, 201 B Nguyen Chi Thanh Street, District 5, HCM City, Viet Nam. Fax: , lengochung@hcm.vnn.vn parasite density in malaria patients treated with artemisinin derivatives was recorded even when the drugs were not found in the blood. This matter may be explained by the post-antiparasitic activity of the artemisinin derivative drugs. Therefore, the efficacy of DHA may be enhanced by an increase of the treatment dose, by the prolongation of the treatment course, or both. We conducted an open randomized comparison between high-dose monotherapy with DHA and a combination of high-dose DHA with mefloquine (MQ) as the treatment for Vietnamese patients with uncomplicated falciparum malaria. PATIENTS AND METHODS Patients and treatment: The study was conducted in two provinces, Binh Thuan and Binh Phuoc, in the southern part of Viet Nam. Binh Thuan Province contains mountains and plains along with a long expanse of seashore. It is considered to be the joining province between the central part and the southern part of Viet Nam. There are some malaria-endemic areas in this province located in the new economic zones and in villages at the foot of mountains. Malaria season often runs from June to December. In this province, the study was conducted at three sites: Bac Binh District Hospital and the Mepu and Song Luy village health care posts. Binh Phuoc Province is located near the border between Viet Nam and Cambodia. This area specializes in rubber plantations and farms for black pepper cultivation. The study was done at the health care post of the 11th rubber farm of the Phu Rieng rubber plantation company. The workers on the plantation and people living in surrounding areas often come to the health 161

2 care post to be checked for malaria when they experience fever. In both provinces, malaria is the main infection disease, with the P. falciparum and P. vivax strains having the same incidence rates. The total duration of the study was 19 months, from January, 2002, until July, The study protocol was reviewed and approved by the Ethical Committee of Cho Ray Hospital, Viet Nam, and was also approved by the WHO/TDR (Tropical Disease Research) Committee. The inclusion criteria were male or female patients with uncomplicated falciparum malaria having a parasitemia density of 1,000/mm 3 or higher but less than 200,000/mm 3, aged 15 years or older, who had freely given written informed consent to the study and agreed to complete the follow-up schedule after treatment. The exclusion criteria were vivax malaria patients, or patients with mixed malaria infection, or pregnant women with falciparum malaria, women during breast feeding period, patients with severe and complicated falciparum malaria, or patients who could not tolerate the oral medication, patients with a history of allergic reaction to MQ or artemisinin derivatives, patients who received artemisinin derivatives in the previous 24 h, or took MQ, tetracycline or doxycycline in the previous 7 days, or quinine in the previous 12 h, and finally patients who refused to give written informed consent. Eligible uncomplicated falciparum malaria patients randomly received one of two study regimens, allocated by randomization code. The random codes were based on the block of 4 with a ratio of 1:1 between the two treatments. Each code was put in a separate envelope, and the envelopes were sealed and numbered. The number of envelopes given to each study site was based on the multiple of block of 4. At the study sites, the randomized treatment codes in the sealed envelopes were distributed to the patients starting from the lowest number. The study size was calculated in order to determine any significantly clinical difference between the two regimens. This calculation was based on the assumption that the recrudescence rate in the DHA-MQ, which is 5%, is about 25% lower than that in the DHA-5 day monotherapy, which is 30%. These recrudescence rates are based on previous studies in the same study areas (12-14) (Hung, L.N. Personal communication). The sample size was 43 cases per study arm, for a total of 86 cases. With the strictly follow-up, the rate of drop-out cases was estimated as 5%. So, the final study size was 90 cases, in which 45 cases for each regimen. The eligible patients received either a DHA-5 day monotherapy regimen with a total dose of 900 mg divided into 300 mg doses on the first 2 days (0 h, 24 h) followed by 100 mg doses on the next 3 consecutive days (48 h, 72 h, and 96 h), or a DHA-MQ combination regimen given as 300 mg DHA at 0 h then followed by another 300 mg DHA and 750 mg MQ at 24 h. If patients vomited within 1 h after drug administration, the full dose of the drug was repeated. DHA capsules (100 mg/capsule) were kindly provided by Dr. F.H. Jansen of the Drafa Pharma, Brussels, Belgium. MQ (Euphaquine ) tablets (250 mg/tablet) were kindly provided by Prof. Kesara Na-Bangchang, Head of the Clinical Pharmacology Department, Thammasat University, Bangkok, Thailand. Clinical procedure: Patients underwent a general physical examination for the evaluation of all baseline data at the time of enrollment in the study. All clinical symptoms such as spleen enlargement, hepatomegaly, anemia, jaundice, and nutritional status as well as functional symptoms such as loss of appetite, fatigue, headache, nausea, and tinnitus, were recorded as baseline signs/symptoms in the patient record forms (hospital treatment forms) or in the case record forms of the study. The progress of treatment as well as the appearance of drug adverse events was evaluated based on a comparison with corresponding baseline signs/symptoms. Body temperature was measured in the axillary region, and then 0.5 C was added. The body temperatures were recorded before drug administration, then followed-up every 8 h until the patient showed a normal temperature ( 37 C) on three consecutive measurements. Pulse and blood pressure were measured every 8 h or 2 times per day according to the working schedules of the study sites. Patients were re-evaluated with a clinical visit at least 2 times per day during the treatment time at the study sites. Patients discharged from the hospital or health care posts only when they completed the treatment and recovered well from all symptoms of malaria, as well as having three consecutive negative blood smears with P. falciparum. All study drugs were given directly to patients by responsible doctors or nurses. At the health post of the 11th Phu Rieng rubber farm, hematology and biochemistry tests were not available. At the Mepu and Song Luy village health posts, only hematology tests for red blood counts and white blood counts with cell differentiation were performed on day 0 (at enrollment time into the trial) and on day 2. At Bac Binh Hospital, hematology tests (complete blood count) and biochemistry tests (serum transaminases, blood bilirubin, blood urease nitrogen, albumin) were performed on days 0 and 2. Blood smear examination: The parasitemia were evaluated on blood smears taken from the tips of patient s fingers. Blood smears were done on admission, then every 8 h after the start of drug administration until three consecutive blood smears that were negative for P. falciparum parasites were confirmed. Then, the blood smears were done daily until the day of discharge. All blood smears were stained with Giemsa and examined by experienced technicians at the health facilities. The parasites were count over 200 white blood cells in the thick blood smear. The parasitemia density was calculated based on the assumption of 8,000 white blood cells per microliter of blood in all patients and expressed as the number of parasites per microliter of blood. The presence of gametocytes in the blood smear was qualitatively recorded as positive or negative. Blood smears were also done when patients returned on days 7, 14, 21, and 28 for the DHA-5 day monotherapy, and two additional times on days 35 and 42 for the DHA-MQ combination regimen. All blood smears of study sites were retained and then checked by microscopic experts of the Malaria Division of Ho Chi Minh City. The results obtained from these experts are considered to be standard values. If there were discrepancies in the parasite density findings between the technicians of the study sites and the experts of Malaria Division of Ho Chi Minh City, the data from the reviewing experts were accepted for the analysis. Patient follow-up: Patients discharged after the treatment was completed and the initial clinical cure was achieved. All patients were requested to be back at the health facilities for reexamination on days 7, 14, 21, and 28 for the DHA monotherapy group and for two additional examinations on days 35 and 42 for the DHA-MQ combination group. On the follow-up days, patients had a general physical examination, blood smears were taken for parasitemia review, then they returned home if their blood smears were negative and they showed no clinical symptoms of malaria. Outcome evaluation: The initial clinical cure was defined 162

3 as the combination of the parasite clearance, the defeverescence and the complete absence of clinical symptoms of malaria. The fever clearance time (FCT) was defined as the time from the beginning of the treatment until the first time of three consecutive normal temperature records ( 37 C). Parasite clearance time (PCT) was defined as the time from the beginning of treatment until the first time of three consecutive negative blood smears for P. falciparum parasite. The 95% PCT and 50% PCT were calculated with the normal logarithmic method. The radical cure or sensitive response of the regimen was defined as initially negative parasitemia by day 7 without any recrudescence up to day 28 for the DHA-5 day monotherapy or day 42 for the DHA-MQ combination regimen. The early RI response was defined as being initially negative for parasitemia then positive again before or on day 14. The late RI response was defined as being positive again for parasitemia after day 14. The RII response was defined as the parasite density reduced by at least 75% (or less than 25% of the initial parasitemia sustained) by 48 h after the treatment, then showing a resurgence without parasite clearance by day 7. The RIII response was defined as no response or parasite density reduced by less than 75% of the initial value by 48 h and not cleared by day 7. Clinical and laboratory adverse effects were recorded if new events occurred in patients after they received the treatment drugs or if the severity of baseline clinical sign(s)/symptoms and/or laboratory findings increased in comparison with those observed before the initiation of treatment. All adverse events were evaluated whether they were related to the study drugs or not. Data management and analysis: At Bac Binh Hospital, the main source documents were the patient record forms (PRFs), including all laboratory records. The source data in the PRFs were then transferred carefully by the study doctors to the case record forms (CRFs) generated for the study. Some data such as body temperature, blood pressure and parasitemia density were directly written on the CRFs. In other health care posts, there were no PRFs; the patient administration books were used as source documents for all patients. The patient data in these books included the ordinal numbers of patient admitted per day, dates of admission, gender, age, patient addresses, blood smear results, treatments given, and dates of discharge. Thus, the CRFs were also used as source documents for other data of the study (patient history, drug treatment monitoring, clinical/laboratory findings, fever monitoring chart, parasitemia follow-up table, etc. The heads of the health care posts were responsible for entering data in the CRFs. The principal investigator was the main monitor for all trial sites with the site visits every month during the trial. All data were managed and analyzed by the Epi-Info Program, version 3.3.2, February, The quantitative data between two regimens were compared using the analysis of variance (ANOVA) if their variances were homogeneous (with Bartlett s test), or by the Kruskal-Wallis test if their variances were not homogeneous. Qualitative data were compared using the chi-square test. The level of significant value, P, was set at 0.05 for all statistical tests. RESULTS Patient characteristics: Ninety-one patients were entered into the trial. Two patients were excluded from the final analysis. One patient was excluded due to having a mixed infection involving P. falciparum and P. vivax parasites, while the other was excluded due to a diagnosis of severe and complicated falciparum malaria. One patient in the DHA-MQ group had high values of blood transaminases (SGOT, 87 IU/L and SGPT, 75 IU/L), and blood bilirubin (6.4 mg/dl) on the day of hospital admission. However, these abnormal biochemistry results had no important clinical relevance, so this case was also considered to be eligible for the final analysis. In total, 89 patients were entered into the final analysis. Forty-five patients were treated with DHA-5 day and 44 with DHA-MQ. Forty-seven cases were treated at the health post of the 11th rubber farm of the Phu Rieng company in Binh Phuoc Province, and 42 cases were treated at 3 health facilities in Binh Thuan Province. There were approximately three times more male patients than female patients (68 versus 21 cases, respectively). The mean age of the patients was 29 years, ranging from 16 to 56 years. Most of the patients lived for more than 1 year in malaria areas (85/89, 95.5%). Thirty-four patients (77%) in each group came to the health facilities after 1-2 malaria crises. There was no difference in the clinical signs/symptoms of malaria between the two treatment groups (Table 1). Table 2 presents the laboratory findings in hematology and biochemistry of patients. There were 3 patients in the DHA-MQ regimen group with red blood cell counts below 3,500,000/ mm 3. Three patients in each group had white blood cell counts Table 1. Baseline clinical characteristics of patients DHA-5 day DHA+MQ (2 days) n = 45 n = 44 Gender: Female/Male 11/34 10/34 Province: Binh Thuan Binh Phuoc Hospital/Health center Bac Binh hospital 8 6 Mepu health center 9 10 Song Luy health center 5 4 Phu Rieng rubber plantation Age (years) Mean ± SD 29.3 ± ±10.1 (range) (16-56) (16-52) Weight (kg) Mean ± SD 49.0 ± ± 5.5 (range) (34-60) (33-63) Height (cm) Mean ± SD ± ± 6.7 (range) ( ) ( ) Living in malaria area (years) No. of cases Mean ± SD 8.3 ± ± 6.5 (range) (1-30) (1-22) No. of malaria crisis before admission 1-2 crisis crisis crisis 3 1 No. of patients with the clinical symptoms (%) Headache 41 (91.1) 43 (97.7) Fatigue 36 (80) 35 (79.5) Anorexia 33 (73.3) 29 (65.9) Arthralgia 9 (20) 8 (18.2) Diarrhea 0 (0.0) 2 (4.5) Nausea 1 (2.2) 5 (11.3) Chill 40 (88.9) 35 (79.5) Hepatomegaly 1 (2.2) 1 (2.3) Splenomegaly 2 (4.4) 4 (9.1) 163

4 Table 2. Baseline laboratory characteristics in blood of patients DHA-5 day DHA+MQ (2 days) n = 45 n = 44 Red blood cells (/mm 3 ) n = 22 n = 20 Mean ± SD 3,981,818 ± 153,177 3,875,000 ± 261,322 (range) (3,800,000-4,400,000) (3,200,000-4,300,000) 3,500,000/mm 3 0 case 3 cases White blood cells (/mm 3 ) n = 22 n = 20 Mean ± SD 6,927 ± 1,189 6,530 ± 1,270 (range) (4,000-9,000) (4,000-8,000) 3,500/mm 3 3 cases 3 cases Neutrocytes (%) n = 22 n = 20 Mean ± SD 61.0 ± ± 8.5 (range) ( ) ( ) SGOT (IU/L) n = 8 n = 6 Mean ± SD 11.1 ± ± 28.5* (range) (3-23) (11-87) SGPT (IU/L) n = 8 n = 6 Mean ± SD 15.3 ± ± 25.5 (range) (6-40) (6-75) Bilirubin (mg/dl) n = 8 n = 6 Mean ± SD 0.99 ± ± 2.19 (range) ( ) ( ) Protein (g/dl) n = 8 n = 6 Mean ± SD 6.9 ± ± 0.6 (range) ( ) ( ) Serum creatinine (mg/dl) n = 8 n = 6 Mean ± SD 0.86 ± ± 0.18 (range) ( ) ( ) Glucose (mg/dl) n = 8 n = 6 Mean ± SD 112 ± ± 35 (range) (83-209) (88-182) *: P = (Mann-Whitney U test), significant difference between two groups. Table 3. Clinical and parasite responses of patients DHA-5 day DHA+MQ (2 days) n = 45 n = 44 Good clinical response 1) Fever clearance time (h) Mean ± SD 23.3 ± ± 19.8 (range) (6-60) (6-99) 6-24 h (cases) h (cases) 9 15 > 48 h (cases) 3 3 Parasite clearance time 100% Mean ± SD 35.3 ± ± 19.2 (range) (12-96) (12-96) Parasite clearance time 95% Mean ± SD 26.6 ± ± 14.6 (range) Parasite clearance time 50% Mean ± SD 8.3 ± ± 5.3 (range) ( ) ( ) Parasitemia response (no., %) Radical cure 33 (73.4) 39 (88.6) Early R1 1 (2.2) 0 Late R1 11 (14.4) 5 (11.4) Radical cure rate by time (no., %) 1) d (100) 44 (100) d (88.89) 43 (97.73) d (75.55) 43 (97.73) d (93.18) d (88.64) Radical cure rate/study sites 2) (%) Bac Binh 5/8 (62.5) 4/6 (66.7) Mepu 9/9 (100) 10/10 (100) Phu Rieng 3) 14/23 (60.8) 22/24 (91.7) Song Luy 5/5 (100) 3/4 (75) 1) : Kaplan Meier curve analysis, Log-Rank = 13.2, P = ) : Data presented as number of cases with radical cure over the total cases. 3) : Significant difference, P = (Chi-square). Fig. 1. Radical cure rate of two regimens, DHA-5 day and DHA-MQ, in uncomplicated falciparum malaria, by follow-up time after receiving treatment. The radical cure rate of DHA-MQ is significantly higher than that of DHA-5 days (P = 0.003). less than 3,500/mm 3. Only the levels of SGOT were found to show a significant difference between the two groups. One patient in the DHA-MQ group with high blood transaminase values caused this difference. There was no difference in the hematology and biochemistry values between the two patient groups. Clinical response: After treatment, all patients recovered quickly from the symptoms of malaria, within 1-3 days. The FCTs were 23.3 ± 13.4 h (mean ± SD; range, 6-60) and 26.2 ± 19.8 h (range, 6-99), respectively, in the DHA-5 day and DHA-MQ regimens (P > 0.05). More than 90% of patients in both groups were free from fever within 48 h after receiving treatment. No RIII or RII responses were found in any of the patients. The PCTs were 35.3 ± 17.4 h (mean ± SD; range, 12-96) and 37.8 ± 19.2 h (range, 12-96), respectively, in the DHA-5 day and DHA-MQ regimens (P > 0.05). No difference in the values of PCT 95% and PCT 50% was found between the two regimens (Table 3). Parasite response: Table 3 presents the details of parasite response during follow-up time between the two regimens. Twelve patients had parasitemia by day 28 in DHA-5 day monotherapy (26.7%) and 5 cases relapsed by day 42 in the DHA-MQ combination regimen (11.4%) (P = 0.07). All recrudesced patients were treated with the current standard combination regimen of CV8 (DHA + piperaquine + primaquine + trimethoprim) or artesunate-7 days. If the comparison was done based on the results as of day 28, the cure rate in the DHA-MQ combination therapy was significantly higher (43/45 cases, 97.7%) than that of the DHA-5 day monotherapy (P = 0.003). The survival analysis of radical cure rate by time showed that the DHA-MQ combination had a significantly higher radical cure rate than the DHA-5 day monotherapy (P = 0.003, Kaplan Meier analysis) (Fig. 1). Stratified by site of study, there was no difference in cure rate between the two regimens except at Phu Rieng health post, where the combination regimen had a higher cure rate of 91.7% compared to that of the DHA monotherapy (60.8%, P = 0.012). DISCUSSION The study showed that DHA was well tolerated with no significant adverse drug reaction. Almost all patients were free from fever and parasitemia within 3 days after the treat- 164

5 ment. The aim of this trial was to determine the efficacy in terms of the radical cure rate of the DHA-5 day monotherapy when a high total dose of 900 mg was used. However, the cure rate of DHA-5 day monotherapy was as low as 73.3% by day 28. When DHA was first used as monotherapy in malaria treatment, it was administered at a low dose (i.e., 240 mg) and over a short course (i.e., 3 days). This low-dose and shortcourse DHA treatment gave a low cure rate of 52% (15). The cure rates were increased to 94 and 98% when the total doses were higher (360 and 480 mg, respectively) and the courses were also longer (5 and 7 days, respectively) in Chinese patients with uncomplicated falciparum malaria (15). A high dose of 600 mg of DHA over 5 days gave cure rates of 80 to 92% in Thailand (16). In Viet Nam, the cure rate of DHA, 600 mg, over 5 days, was 87.5% in (Hung, L.N. Personal communication). All of these studies suggest that high doses of DHA may increase the cure rate and prevent the occurrence of drug resistance. However, the use of high doses of DHA seems unsuccessful in the present clinical trial. There are many possible reasons for this failure, such as: (i) the dose was not high enough to give good efficacy; (ii) there was a difference in the bioequivalence of DHA drug in this trial, i.e., the low bioavailability factor; (iii) the self-reduction of drug concentrations for DHA may be due to enzyme induction, which could be more important than the increase of the treatment dose; and (iv) the beginning emergence of parasite resistance against DHA or other artemisinin derivatives due to the long-term use of artemisinin and its derivatives in Viet Nam. There have been very few pharmacokinetic studies on the oral forms of DHA using the same method of high performance liquid chromatography with an electrochemical detector (17-19). In this study, we used the DHA produced by Drafa Pharma. Up to now, there has been no bioequivalence study on the oral DHA formulation. However, it seems that there was no difference in the pharmacokinetic parameters between the two DHA formulations, Arenco-nv (of Drafa Pharma) and Cotecxin (China), in Thai and Vietnamese healthy volunteers, respectively (17,19). The t max and t 1/2z were roughly similar between the two formulations, i.e., around 1.5 and 2 h, respectively. The C max (maximum concentration) and AUC (area unclear the concentration-time curve) of DHA were also comparable between Thai and Vietnamese healthy subjects. Thus, the effect of bioequivalence on the efficacy of DHA in the treatment of patients in this study was considered to be negligible. The maximum in vitro inhibition concentration (IC 99 ) of DHA was reported to be 7.5 ng/ml (20). The maximum concentrations (C max ) of the oral DHA formulation (Cotecxin) at doses of 60 mg and 240 mg in Vietnamese malaria patients were 490 ± 200 ng/ml and 862 ng (345-2,280), respectively (21) (Hung, L.N. Personal communication). With the assumption of the same bioequivalence between the two formulations of DHA, Cotecxin tablets and DHA capsules, the C max of DHA (300 mg) on the first day of treatment in this trial could be estimated to be equal to or higher than that in the two above trials. The parasitemia continuously declined even when the DHA concentration was lower than the maximum in vitro IC 99. Thus, the minimum parasiticidal plasma concentration (MPC) of DHA could be lower than the maximum in vitro IC 99 (22). This raises the issue of the post-antiparasitic efficacy (PAE) of DHA. In the field of antibiotic drugs, those having postantibiotic efficacy are classified as having dose-dependent antibiotic activity. Such antibiotics, i.e., gentamycin and amikacin, are required to be given once per day, with a higher dose having higher antibiotic activity. It is appropriate that, in clinical experience, most artemisinin derivatives (including DHA) are often given as once daily dose. In this trial, when higher doses of DHA were used, the higher C max concentrations could be reached, but the radical cure rate was lower. So the concept of the PAE of DHA may not be sufficient to explain the failure of the treatment. This means that besides the dose-dependent antiparasitic activity, the time-dependent antiparasitic characteristics are also important for killing all residual parasites that escape the PAE action of DHA. The time-dependent induction was suggested as the cause for the reduction of DHA concentrations in the 5 day monotherapy course in patients (11). In response to this problem, a higher treatment dose or/and a longer duration of treatment (i.e., 7 days) may be required in DHA monotherapy. In laboratory research, artemisinin-resistant strains of P. falciparum parasites have been developed (23). The sensitivities of the parasite strains isolated from patients vary greatly in in vitro tests, but there is no proof that this sensitive variation is related to the clinical failure, may be due to the difference in the capacity for survival of drug-resistant parasites in the culture medium (20,24). However, WHO in 1997, reported some reduced susceptibility of falciparum parasites to artemisinin in Yunnan Province, in the border areas between China, Lao PDR and Myanmar, due to migration and an increase in self-treatment in this area (25). All of the risk factors that contributed to the emergence of resistance to other antimalarial drugs may also play a role in the emergence of artemisinin resistance. In this trial, the sensitivity of DHA used alone was lower than the sensitivities reported in previous studies (12,14,26) (Hung, L.N. Personal communication). This finding may suggest a need to follow-up the signs of the emergence of artemisinin resistance in Plasmodium parasites in Viet Nam. This study again showed that the combination therapy of DHA-MQ had a good treatment result over 42 days, even in the malaria transmission areas where recrudescence and re-infection cannot be differentiated. The combination of artemisinin derivatives and MQ provides some advantages in the treatment because: (i) artemisinin derivatives show in vitro synergism with both MQ and quinine (30); (ii) the quick antimalarial killing action of artemisinin derivatives greatly reduces the biomass of the parasites, leaving a small amount of parasites to be cleared by the second drug (i.e., MQ), and thus causing a reduction in the resistance of parasites to MQ (27,29); (iii) inversely, MQ in the combination regimen also has killing action on the few remaining parasites and protects artemisinin derivatives from drug resistance of parasites (27,29); and (iv) artemisinin derivatives have the additional benefit of reducing the production of gametocytes due to their rapid clearance of the biomass of asexual parasites and their activity against the early stage of sexual forms of parasites (28). The inhibition of gametocytogenesis may further help to delay the emergence of drug resistance. The low response in terms of the radical cure rate of DHA monotherapy, even when the total dose was increased, indicates that it should not use artemisinin derivatives alone in P. falciparum malaria treatment. The combination regimens involving artemisinin derivatives with other long-acting antimalarials, such as MQ, were significantly more effective. The study showed that the radical cure rate of high-dose 165

6 monotherapy of DHA over 5 days, with a total dose of 900 mg, was not increased. Inversely, the cure rate of the combination regimen, DHA + MQ, with a high dose of DHA of 300 mg, over a 2-day treatment course, was stable at 88.7% on day 42 after the initial day of treatment. The study also found that there were no adverse events even when DHA was administered at a high dose. Thus, the artemisinin combining therapies (ACTs) may be better candidates for the treatment of patients with uncomplicated falciparum malaria. ACT can be considered as an important tool in combating drug resistance in the treatment of falciparum malaria. ACKNOWLEDGMENTS Le Thi Diem Thuy is in receipt of WHO/TDR fellowship for PhD degree in Clinical Pharmacology. We thank the staff members of the Research and Training Department, Cho Ray Hospital, the Faculty of Allied Health Sciences, Thammasat University and the Lam Dong II Hospital who helped us in this study. REFERENCES 1. 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