AJTMH ASTMH MEMBERSHIP INFORMATION: astmh@astmh.org
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am. J. Trop. Med. Hyg., 71(6), 2004, pp. 723-731
Copyright © 2004 by The American Society of Tropical Medicine and Hygiene

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by LI, Q.
Right arrow Articles by MILHOUS, W. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by LI, Q.
Right arrow Articles by MILHOUS, W. K.
Related Collections
Right arrow Malaria

PHARMACOKINETIC INVESTIGATION ON THE THERAPEUTIC POTENTIAL OF ARTEMOTIL (ß-ARTEETHER) IN THAI PATIENTS WITH SEVERE PLASMODIUM FALCIPARUM MALARIA

QIGUI LI, CHARLES B. LUGT, SORNCHAI LOOAREESUWAN, SRIVICHA KRUDSOOD, POLRAT WILAIRATANA, SUPARP VANNAPHAN, KOBSIRI CHALEARMRULT, AND WILBUR K. MILHOUS
Division of Experimental Therapeutics, Walter Reed Army Institute of Research, Silver Spring, Maryland; ARTECEF BV, Maarssen, The Netherlands; Hospital for Tropical Diseases, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand


ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pharmacokinetic data were obtained to evaluate the therapeutic potential of Artemotil (ß-arteether) in 56 Thai patients with severe Plasmodium falciparum malaria. Intramuscular administration was given at 1) a low dose of 3.2 mg/kg on day 0 and 1.6 mg/kg/day on days 1–4 and 2) a high dose of 4.8 mg/kg on day 0 at 0 hours, 1.6 mg/kg at 6 hours, and 1.6 mg/kg/day on days 1–4. Cmax values of 63.7 ng/mL at 6.1 hours and 140.8 ng/mL at 5.7 hours were reached in low-dose and high-dose patients, respectively. Drug concentrations decreased slowly with half-lives of 12.5–22.4 hours on day 0 and 31.6–40.7 hours on day 4 for both dosage regimens. Although the maintaining dosage on the last day was much lower than the loading dose on day 0, the area under the curve (AUC) and Cmax on day 4 were significantly increased (2.85–4.55 fold), suggesting drug accumulation in the blood. Dihydroartemisinin (DHA), an active metabolite of Artemotil, was detected in most patients. The mean ratios of DHA and Artemotil were 0.16–0.19 in both dosage regimens for the entire study period. Similar to previous reports, all patients showed a slow response to treatment with mean values of 77.2 hours for the fever clearance time (FCT) and 75.8 hours for the parasite clearance time (PCT) (low dose) and 70.1 hours for the FCT and 64.4 hours for the PCT (high dose). Interestingly, a very rapid response to the treatment was exhibited in patient 151, with an FCT of 4 hours and a PCT of 36 hours, with different pharmacokinetic data from others on day 0. The patient had a very high Cmax (2,407 ng/mL) and AUC (12,259 ng·hr/mL) values without an intramuscular absorption phase on the first day. These values were approximately 21.9 (Cmax) and 2.6 (AUC) times higher than in other patients; this patient may have been to be injected through a vessel at first dosing. In conclusion, the patients treated with the high dosage regimen had higher AUC values and higher antimalarial efficiency (cure rate = 48%) than the low-dose subjects (cure rate = 23%). Despite the high accumulation and longer exposure time (9–11 days) when compared with other artemisinin agents, due to the slow prolonged absorption of Artemotil from injection sites, the two dosage regimens did not show a better therapeutic effects than other artemisinin drugs, including {alpha}/ß-arteether dissolved in peanut oil used in Indian patients.


INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In 1985, the World Health Organization, the Walter Reed Army Institute of Research, and ARTECEF BV developed the ether derivative Artemotil (ß-arteether) in sesame oil as an injectable formulation of artemisinin compounds.1 The first test of Artemotil in humans in studies of safety, tolerance, and pharmacokinetics in healthy men was in 1993, where the drug was given in single and multiple doses. At those doses and durations the drug was well tolerated.2

Phase II clinical trials of injectable Artemotil were initiated in 1993 in adult patients with Plasmodium falciparum malaria in The Netherlands and Thailand. Studies were initiated in patients with non-severe malaria to establish an optimal dosage regimen and to characterize the rapidity of the clinical response. In 1994, subjects were given Artemotil intramuscularly at a starting dose of 3.2 mg/kg, followed by 1.6 mg/kg for four days for the treatment of uncomplicated P. falciparum malaria. The regimen proved safe and had potent antimalarial efficacy, reducing parasitemia from the peripheral blood at a low dosage; similar to artemether (AM), which reduced parasitemia by 50–90%.3

In a second trial, 200 patients with severe P. falciparum malaria were studied between July 1995 and June 1997. The objective of the study was to compare the efficacy of intramuscular Artemotil to AM in patients with severe P. falciparum malaria. All patients were treated by one of the three regimens: 1) low dose of Artemotil: 3.2 mg/kg on day 0 and 1.6 mg/kg of on days 1–4, 2) low dose of AM: 3.2 mg/kg on day 0 and 1.6 mg/kg on days 1–4, or 3) high dose of Artemotil: 4.8 mg/kg on day 0, 1.6 mg/kg six hours later, and 1.6 mg/kg/day on days 1–4. The results of the study showed that the two low-dose treatments had survival percentages within the expected range: Artemotil = 83.6% and AM = 88.7%. This difference was not statistically significant. A higher initial parasite count, renal failure, and injection into the thigh resulted in a significantly lower chance of survival. Parasite clearance time (PCT) did not differ between the two treatments. Comparable means were found for fever clearance time (FCT), approximately 80 hours, which was confirmed by statistical evaluation. Coma recovery time for both low dose groups was approximately 40 hours with no significant differences found for Artemotil compared with AM in high-dose treatments.3

The phase III study was reported in 1998 from two of the three centers that studied this drug in African children with cerebral malaria. Ninety-two children were studied: 48 received Artemotil and 44 received quinine. No significant differences in survival, coma resolution time, neurologic sequelae, PCT, and fever resolution time were seen between the two regimens. Rates for negative malaria smears one month after therapy were similar in both groups. Artemotil was a well-tolerated drug in the 48 patients in this study. It appears to be at least therapeutically equivalent to quinine for the treatment of pediatric cerebral malaria and has the advantage intramuscular dosing once a day for only five days.4 A comparison study between intramuscular Artemotil and intravenous quinine has been evaluated in 102 children from Cameroon. Artemotil with sesame oil has been shown to be safe and therapeutically as effective as quinine for the treatment of cerebral malaria in children.5

Intramuscular administration of Artemotil in sesame oil yielded a low blood concentration and a long elimination half-life. It was expected that the half-life of Artemotil with sesame oil and its more lipophilic properties would be longer than with other artemisinin drugs, thus favoring accumulation in blood and brain tissue as advantages in treating treat severe and cerebral malaria.6 The comparison of the therapeutic potential of Artemotil with other artemisinin drugs in humans for 3–5-day treatments is summarized in Table 1Go.3,6–27 The rates of recrudescence, fever, and parasite clearances are traditional evaluations for the efficacy of the antimalarial drugs in humans. These three parameters showed that the therapeutic potential of intramuscular Artemotil is not superior to that of oral dihydroartemisinin (DHA), oral and intravenous artesunate (AS), intramuscular AM, or even intramuscular {alpha}/ ß-arteether formulated with peanut oil. However, in vitro studies reported the relative antimalarial potency of artemisinin compounds as 1 (artemisinin) < 1.2–1.5 (artelinic acid) < 1.9–2.3 (artemether) < 2.2–2.6 (ß-arteether) < 3.9–4.8 (artesunate) < 3.6–5.8 (dihydroartemisinin) with culture parasites of P. falciparum.28–30 Since ß-arteether is not any less potent than artemisinin drugs in vitro, why is the antimalarial potential of Artemotil inferior to other artemisinin compounds in clinical use?


View this table:
[in this window]
[in a new window]
 
TABLE 1
Summary of clinical trials of artemisinin derivatives monotherapy in treatment of patients with severe/complicated malaria (all data cited from published literature)*
 
The therapeutic potential with the optimal dosing regimens for Artemotil has been evaluated in previously described human studies,31 yet efficacy has not been projected from other artemisinin drugs and quinine. Pharmacokinetic measurements can be a useful tool in obtaining more insight into the therapeutic potency of a treatment regimen for Artemotil and would help to evaluate clinical effects. In the present study, the pharmacokinetics of Artemotil and its active metabolite DHA were evaluated to explain the basis of the low efficacy in clinical trials of Artemotil after intramuscular administration.


PATIENTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects. This trial was an open label, randomized, comparative, and single-center study in adult patients. The study protocol and the informed consent forms were reviewed and approved by the Institutional Ethics Committee and the Ethics Committee of Mahidol University. The antimalarial efficacy and tolerability of Artemotil was compared in low-dose and high-dose regimens in severe malaria patients with naturally acquired P. falciparum infection. The study focused on the pharmacokinetic properties of Artemotil and its demethylated metabolite DHA. Adult patients (age = 16–60 years) with symptomatic, previously untreated P. falciparum infection were included in the criteria. Patients in the study included those with signs of severe malaria, cerebral infection, hyperparasitemia, and renal failure. They were randomized and treated with either a low-dose (n = 15) or high-dose regimen (n = 55). These 70 patients were treated with intramuscular injections in the gluteal region once a day for five days (twice on day 1 for the high-dose regimen).

Treatment and observation. Sornchai Looareesuwan (Bangkok Hospital for Tropical Diseases, Bangkok, Thailand) manufactured the trial medication. Medication was administered intramuscularly in the gluteal region on alternating sides for five days at the low dose of 3.2 mg/kg on day 0 and 1.6 mg/kg/day on days 1–4 in 15 patients and at the high dose of 4.8 mg/kg on day 0, 1.6 mg/kg six hours after the initial injection, and 1.6 mg/kg/day on days 1–4 in 55 patients. Randomization was in blocks of five to include four high-dose Artemotil patients and one normal-dose (low dose) Artemotil patient. Clinical outcomes after a change in treatment, e.g., to intravenous AS, were also recorded in the coma recovery time.

Clinical observations were recorded at the clinic from -12 hours up to 28 days after the first drug injection or alternatively from -12 hours up to day 7 with visits on days 14, 21, and 28. Blood lactate concentrations were measured every six hours (in the same samples as blood glucose) until 24 hours after the first dose. Full physical and neurologic examinations were given before the first injection and twice a day until the patient had cleared fever, parasitemia, and coma and on days 7, 14, 21, and 28. Blood films and oral body temperature were obtained every six hours until films were negative and the temperature was < 37.5°C; thereafter, films and temperature were obtained once a day to determine recrudescence failure.

Plasma sample preparation. Blood samples (5.0 mL) for pharmacokinetic analysis were drawn repeatedly via an indwelling catheter with a heparin lock in all patients. Blood was collected into sterile plastic syringes, transferred immediately into heparinized tubes, and centrifuged at 3,000 rpm for 15 minutes at 4°C. Plasma samples were aliquoted into two glass tubes (1 mL each) and stored in dry ice or an electric freezer at –80°C. Duplicate samples were transported by thermo container containing dry ice to the laboratory at the Walter Reed Army Institute of Research in Washington, DC. On delivery, samples were inventoried and stored at –80°C until analysis could be performed. Quality control (QC) and standard curve (SC) samples were prepared in the blank plasma as close as possible to the date of sample receipt and were stored under the same conditions as the clinical samples. The QC and SC samples consisted of Artemotil and DHA in human blank plasma at different concentrations (0–1,000 ng/ mL). Six QC and 10 SC samples were assayed with each set of patient samples; they were randomly placed among clinical samples for each assay run. Six QC samples were assayed consecutively within an analysis.

A liquid-liquid extraction method was used for sample preparation as previously described.32 Artemotil and DHA concentrations were measured by reverse-phase high-performance liquid chromatography with electrochemical detection (HPLC-ED).

Analysis by HPLC-ECD. The HPLC-ECD was performed using a model BAS 200 liquid chromatography system (Bio-analytical Systems, West Lafayette, IN). This system has three mobile phase reservoirs, solenoid proportioning valves, a dual piston pump, a pulse dampener, a column and detector oven, dual thin-layer electrodes with an Ag/AgCI reference electrode, and a Rheodyne injector for manual injection, which was modified for reductive work. The system is also equipped for mobile phase heating and sparring. The HPLC-ECD was performed using minor modifications of the method of Li and others.33

Pharmacokinetic data evaluation. Extensive pharmacokinetic analysis was carried out on the first 60 patients in both regimens of dose treatment. Venous blood samples were taken through an antecubital catheter at 0, 2, 4, 8, 12, 18, 24, 48, 72, 96, 108, 120, 144, 168, and 216 hours. To determine the pharmacokinetic parameters of Artemotil and the concentration-time data of Artemotil as well as the metabolite DHA, samples collected during first day were fitted to a one-compartment model. On the last day, dosing was fitted to a two-compartment open model using a nonlinear, extended, least-square fitting procedure (WinNonlin 3.1; Scientific Consulting, Inc. Apex, NC). The area under the curve (AUC) for the drug was determined by the linear trapezoidal rule with extrapolation to infinity based on the concentration of the last time point divided by the terminal rate constant. Extrapolations to time zero were done using zero concentration for intragastric dosing and using C0 values determined from the two-compartment model equation at time zero by intravenous route. Mean clearance rate (CL) was determined by dividing the dose by the AUCinf for intravenous injection. Mean residence time (MRT) was determined by dividing the area under the first moment curve (AUMC) by the AUC. The volume of distribution at steady state (Vss) was calculated as the product of CL and MRT. The conversion ratio of DAH/Artemotil was calculated by AUCDHA/AUCArtemotil. Statistical analysis was performed with the statistical software Excel® (Microsoft, Redmond, WA) using a Student’s t-test for dependent samples to compare means of paired samples within one individual.


RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In this trial, 70 patients were enrolled and randomly assigned into the low-dose and high-dose regimen groups. Following treatment, 1–2 patients in each dose group dropped out of the study for reasons unrelated to drug treatment or side effects. Thirteen additional patients were recruited because some of original patients were not evaluable; their blood samples were not collected. All patients had efficacy evaluations; only 56 patients had the full pharmacokinetic analysis done (Table 2Go).


View this table:
[in this window]
[in a new window]
 
TABLE 2
Efficacy and pharmacokinetic parameters of ß-arteether with sesame oil (Artemotil) following im administration daily for five days at the different dosage regimens (low and high) in 56 Thai patients with severe Plasmodium falciparum malaria*
 
Evaluation of efficacy. The initial parasitemia in 11 patients from the low-dose group had a mean value of 436,633 asexual stages/µL, with a range of 25,050–961,350/µL. Parasitemia in 43 patients from the high-dose group had a mean value of 378,045 asexual stages/µL, with a range of 14,640–1,078,440/µL. More than 200,000 parasites/µL (5% parasitemia in red blood cells) are defined as hyperparasitemia. Sixteen of the 70 patients were classified as having cerebral malaria, i.e., having a Glasgow Coma Score (GCS) ≤ 8. Eight of the 70 patients had acute renal failure and required hemodialysis. Another 27 of these 70 patients had anemia and required blood transfusions.

All patients showed a slow initial response (mean PCT = 75.8 hours, mean FCT = 77.2 hours in low-dose patients, and mean PCT = 64.4 hours, mean FCT = 70.1 hours in high-dose patients). One patient (151), who likely received intravenous drug administration, showed a rapid initial response (PCT = 36.0 hours, FCT = 4.0 hours). The low and high doses of Artemotil did not clear the hyperparasitemia completely and resulted in a high recrudescence of 77% in the low-dose patients and 46% in high-dose patients. All recrudescence patients showed recrudescence during follow-up on days 17–21 (Table 2Go).

Evaluation of toxicity. The evaluation of adverse experiences, injection site inspection, electrocardiographic recordings, neurologic examinations, and clinical laboratory tests did not show abnormalities due to any of the two study medications. Clinical observations were recorded at the clinic from –12 hours up to 28 days after the first drug injection or alternatively from –12 hours up to day 7 with visits on days 14, 21, and 28.

Stability of plasma samples. Quality control showed frozen samples of Artemotil in plasma to be stable. The concentration of Artemotil did not change in the first nine months while stored at –80°C. Artemotil levels were slightly lower (1.4–3.9%) when compared with freshly prepared samples after one year at –80°C. The Artemotil concentration of frozen samples was lower (6.6–13.2%) when stored at –80°C for two years. Dihydroartemisinin is less stable than Artemotil in the frozen plasma; its half-life of stability was 14.5 months at –80°C. In the present study, the QC and SC samples were prepared shortly before the date of sample receipt and stored frozen under the same conditions as the clinical samples. The assay quantitation of clinical samples of Artemotil and DHA were not affected by long-term storage.

Pharamcokinetic analysis of Artemotil. The data sets obtained with the pharmacokinetic modeling program were used for the daily dosing interval fitting in patients receiving the low-dose regimen, for the twice dosing interval fitting on day 0 (0 and 6 hours), and for the daily dosing interval fitting on days 1–4 in the high-dose subjects. Good graphic fits of the measured plasma concentration curves obtained for both regimens, indicating that the procedure performed well in estimating the pharmacokinetic parameters. Low inter-parameter correlation and small confidence intervals were obtained with good coefficients of determination (r2 = 0.934–0.971).

The individual levels of Artemotil and the mean plasma concentrations of DHA are shown in Figure 1Go. The main pharmacokinetic parameters following low- and high-dose administrations are shown in Table 3Go. The maintaining intramuscular dose of Artemotil on day 4 was half and one-fourth of that on day 0 (loading dose) in the low-dose and high-dose groups, respectively. However, the plasma concentration of Artemotil on day 4 was three times higher than those on day 0 in both dose groups. Drug accumulations of Artemotil and DHA in the blood were evident. Due to daily increases of the drug levels, the steady state plasma concentrations of Artemotil and DHA were not reached until after the final dose (96 hours).



View larger version (23K):
[in this window]
[in a new window]
  		    FIGURE 1. Pharmacokinetic profiles of Artemotil measured by high-performance liquid chromatography with reductive electrochemical detection (open circles), mean computer fitted curves (solid line), and mean concentration (dashed line) of dihydroartemisinin (DHA), a metabolite of Artemotil, following intramuscular injection of Artemotil with sesame oil at the low dose of 3.2 mg/kg on day 0 and 1.6 mg/kg/day on days 1–4 (top, n = 11) and at the high dose of 4.8 mg/kg on day 0, 1.6 mg/kg at six hours, and 1.6 mg/kg/day on days 1–4 (bottom, n = 44) in the severe malaria patients infected with Plasmodium falciparum in Bangkok, Thailand. Conc. = concentration.

 

View this table:
[in this window]
[in a new window]
 
TABLE 3
Main pharmacokinetic parameters of ß-arteether with sesame oil (Artemotil) following multiple im administration of a low-dosage regimen (3.2 mg/kg on day 0 and 1.6 mg/kg on days 1–4, n = 11) and a high-dosage regimen (4.8 mg/kg on day 0 and 1.6 mg/kg at 6, 24, 48, 72, and 96 hours, n = 44) in Thai patients with severe Plasmodium falciparum malaria*
 
The Cmax values of Artemotil increased from 63.7 ng/mL on day 0 to 110.1 ng/mL on day 4 in the low-dose group and from 140.8 ng/mL on day 0 to 241.2 ng/mL on day 4 in the high-dose group. The AUC data increased from 2,521 ng·hr/mL on day 0 to 8,409 ng·hr/mL on day 4 in the low-dose group and from 4,702 ng·hr/mL on day 0 to 9,484 ng·hr/mL on day 4 in the high-dose group. The elimination half-life of Artemotil increased from 12.5 hours on day 0 to 40.7 hours on day 4 in the low-dose group and from 22.4 hours on day 0 to 31.6 hours on day 4 in the high-dose group (Table 3Go). The elimination half-lives calculated for artemotil on day 4 are not true elimination half-lives. Prolonged absorption of artemotil from sesame oil will extend the plasma concentrations and make the elimination half-life appear longer than the true elimination half-life.

Pharmacokinetic analysis of DHA. The peak plasma concentration of DHA, an active metabolite of Artemotil, was much lower than its parent drug (Figures 1Go and 2Go). The pharmacokinetic parameters showed that the AUC value of Artemotil during the entire treatment period was 5.3-fold higher than that of DHA in low-dose patients and 6.2-fold higher in high-dose patients (Table 3Go). The ratio of AUCDHA to AUCArtemotil was 0.19 ± 0.14 (mean ± SD) in the low-dose group and 0.16 ± 0.07 in the high-dose group during the entire treatment period (Table 3Go).



View larger version (10K):
[in this window]
[in a new window]
  		    FIGURE 2. Pharmacokinetic profiles of Artemotil measured by high-performance liquid chromatography with reductive electrochemical detection (open circles), mean computer fitted curves (solid line), and mean concentration (dashed line) of dihydroartemisinin (DHA), an active metabolite of Artemotil, following administration of Artemotil with sesame oil at the high dose of 4.8 mg/kg on day 0, 1.6 mg/kg at six hours, and 1.6 mg/kg/ day on days 1–4 (n = 42) in the patient 151 who seemed to be incidentally injected with Artemotil through a vessel at first dosing without an intramuscular absorption phase for both Artemotil and DHA. Conc. = concentration.

 
Similar to Artemotil, accumulation of DHA was also found in this clinical trial following the five-day treatments (Figure 1Go). The Cmax increased 1.6-fold from day 0 (12.7 ng/mL) to day 4 (20.3 ng/mL) in the low-dose group and 3.3-fold in the high-dose group (Table 3Go). On day 4, AUC values were 1,273 ng·hr/mL in the low-dose group (a 14.7-fold increase) and 1,411 ng·hr/ml in high-dose group (a 10.7-fold increase). The half-lives of DHA also increased from 14.7–16.0 hours on day 0 to 23.8–24.4 hours on day 4 in the both dose groups (Table 4Go). The MRT of DHA (33.2–34.0 hours) was shorter than that of Artemotil (44.9–61.4 hours) on day 4, suggesting that DHA in the human body does not last longer that the parent drug Artemotil (Tables 3Go and 4Go).


View this table:
[in this window]
[in a new window]
 
TABLE 4
Pharmacokinetic parameters of dihydroartemisinin, an active me-tabolite of Artemotil, following multiple im administration of a low-dose regimen (3.2 mg/kg on day 0 and 1.6 mg/kg on days 1–4) and a high-dosage regimen (4.8 mg/kg on day 0 and 1.6 mg/kg at 6, 24, 48, 72, and 96 hours) in Thai patients with severe Plasmodium falciparum malaria*
 
Pharmacokinetic analysis of patient 151. Profiles of concentration versus time for Artemotil and DHA in patient 151 from the high-dose group are in Figure 2Go, indicating that his pharmacokinetic profiles were different from other patients. Patient 151 at first dosing seemed to be incidentally injected through a vessel and showed an intravenous injection profile with very high Cmax concentration right after injection and a rapid distribution phase. Artemotil and DHA plasma concentrations decreased significantly after first dosing with an elimination t1/2 of 1.1 hours in the first six hours. After the second intramuscular injection at the sixth hour, the elimination t1/2 of Artemotil was immediately increased to 9.35 hours as a daily half-life (Table 3Go). The Cmax (2,407 ng/mL) was approximately 22-fold higher in patient 151 than the mean value (110 ng/mL) reported in other high-dose patients. The AUC value (12,259 ng·hr/mL) was 2.6 times higher than the mean value (7,403 ng·hr/mL) reported in other subjects in the high-dose group. The results were also reported for increasing DHA levels in patient 151.


DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study was conducted to evaluate the efficacy of Artemotil using pharmacokinetic data. After multiple intramuscular dosings, accumulations and longer half-lives of Artemotil were observed. Artemotil and its active metabolite DHA were assayed in each sample from 56 patients who received intramuscular injections of Artemotil in low (3.2 and 1.6 x 4, total = 9.6 mg/kg) and high (4.8 and 1.6 x 5, total = 12.8 mg/kg) doses. Plasma drug concentrations increased more than 3–4 times in both the low-dose and high-dose patients after five days of dosing, even though the maintaining doses were half that of the loading dose in the low-dose group and one-fourth that of the loading dose in the high-dose group.

The data in this accumulation study confirm and extend that of earlier studies in humans and animal species following multiple intramuscular administrations of Artemotil or AM with sesame oil.2,34–37 A rat study demonstrated that the absorption of Artemotil from muscle (injection site) was incomplete on the first day and the bioavailability of Artemotil was 23.4% following single intramuscular administration. At 24 hours after dosing, up to 38% of the total single dose of Artemotil remained at the injection site; at 48 hours, 22% of the total single dose remained at the injection site.9 Based on the calculated half-life (26.3 hours) of the slow absorption phase in muscle, a single Artemotil dose (25 mg/kg) would be completely absorbed in 6–7 days. Acute toxicity data has shown that animals receiving a single high dose of Artemotil in sesame oil died between days 5 and 11.35 The data suggests that the low bioavailability of Artemotil after intramuscularly administration, as well as the delayed onset of toxicity and death in rats, is due to slow and prolonged absorption.

In human studies, no plasma accumulation has been found for artemisinin drugs by other administration routes. Four artemisinin drugs (artemisinin, AS, AM, and DHA) have shown a decreasing concentration in plasma during multiple oral treatments in malaria patients and healthy subjects. When comparing the final dose day to day 0 in these studies, the Cmax and AUC values were markedly reduced from one-third to one-seventh. The decrease in drug exposure during treatment was not disease-related since the pharmacokinetics of artemisinin on day 0 was similar to that reported in healthy subjects.38–43 It is suggested that artemisinin drugs may induce CYP2C19 in an increase of metabolic capacity and undergo auto-induction of the first-pass effect; this may result in a decrease in bioavailability after repeated doses.42,44

In vitro studies have addressed parasite viability after exposure to artemisinin; killing or suppressive effects of the compound in relation to its concentrations and exposure time were assessed. Concentrations of 10–6-10–5 M (280–2,800 ng/ mL) artemisinin showed an effect after only three hours of drug exposure, whereas 10–7 M (28 ng/mL) artemisinin required 24 hours of exposure for a killing effect. At concentrations of 10–8 M (2.8 ng/mL), artemisinin had no appreciable effect on the parasites.45 The low-dose range would not be selected in human treatments due to the very short half-life of artemisinin drugs; as described previously, the longer exposure time at low blood concentrations was more likely to cause fatal neurotoxicity than higher levels of exposure.46 Longer exposure times and low blood levels did not increase efficacy. Longer half-lived Artemotil (12–42 hours) did not increase efficacy. In treating patients with severe and complicated malaria, patient survival is the most important factor. A curative effect is best achieved by switching off the vital functions of the parasite as soon as possible.47 There is no evidence that rapid parasite killing is harmful to the patient, and the therapeutic blood drug concentration should be as high as possible without risking toxicity.48

During the Artemotil clinical trial, patient 151 inadvertently obtained a full or fractional intravenous injection with the intramuscularly formulation in the first treatment. The pharmacokinetic data for this patient showed an intravenous concentration versus time profile and extremely high drug level in plasma (Figure 2Go). Compared with the mean values of other patients who were in same regimen group, the Cmax was 22-fold higher in this patient than in the other 44 subjects. The AUC of patient 151 was 2.6 times higher than that of other patients on day 0. Due to high drug levels, this patient obtained very successful clinical therapy with a PCT of 36 hours and an FCT of 4 hours (which are close to the therapeutic potency of an artesunate injection) compared with other subjects (PCT = 64 hours and FCT = 70 hours). If an improved formulation for intramuscular Artemotil showed more rapid and reliable absorption to the extent seen in patient 151, the therapeutic potential of Artemotil would be much improved and at least as good as other artemisinin drugs.

Another reason for the poor efficacy of Artemotil could be due to the extremely high-count parasitemia in the patients in this study. Mean parasitemias of 436,633 and 378,045/µL were detected in the low-dose and high-dose patients, respectively. These were approximately two times higher than the numbers for hyperparasitemia (> 200,000 parasites/µL or > 5% infected red blood cells) in patients who were also unable to swallow tablets, with coma, anemia, and/or renal failure as the indication of severe malaria.49 The low drug concentration and longer exposure time of Artemotil with sesame oil did not seem to be suitable for the severe malaria patients. Previous pharmacokinetic data demonstrated that the plasma concentration of AM was more than three-fold higher than that of ß-arteether in rats at a same dose level; both drugs were formulated in sesame oil.33 Similar data that showed that Artemotil has a slower rate of absorption than AM was reported in humans.45 It may be that AM has less lipophilic properties than Artemotil (ß-arteether), favoring absorption from muscle. The better efficacy of {alpha}/ß-arteether was found in the peanut oil formulation from India, suggesting that peanut oil may result in easier absorption of this drug in the humans. Further efficacy studies need to be done on the new intramuscular formulation of Artemotil.

Sesame oil and cremophore were used to evaluate the efficacy and pharmacokinetics of Artemotil in two different intramuscular vehicles in the rat.46 Data showed that the Cmax was 13-fold higher after using 25 mg/kg of arteether (AE) with cremophore (AECM) than after using Artemotil at same dose level on day 0. The bioavailability of AECM following dosing was 74.5 ± 12.1% (mean ± SD) (3.7-fold higher than Artemotil). The high bioavailability or the high exposure level contributed to a marked increase of the efficacy in mice. When AECM and Artemotil were administered intramuscularly once a day for three consecutive days in P. berghei-infected mice, the cure dose in 50% of the animals (CD50) was 34.1 (95% confidence interval [CI] = 13.3–66.9) mg/kg for Artemotil and 14.2 (95% CI = 6.8–21.5) mg/kg for AECM. The data confirm that changing the formulation has the ability to change the efficacy of AE.46

Intramuscular Artemotil has the advantage of simple application and fewer undesirable side effects in cases where the patient is not able to retain food. In particular, treatment with intramuscular Artemotil can save time and lives in small children living in areas poorly served by health services. Also, Artemotil has the lowest possible cost and will be made available to developing malaria-endemic countries.37 However, the recrudescence rate is too high (47–71%) compared with other antimalarial regimens for the treatment of severe malaria in these regions. Mefloquine reduces the recrudescence rate from 24% to 5% with intravenous artesunate, from 45% to 20% with intramuscular artemether, and quinine and tetracycline had a recrudescence rate of 4%.50 The fact that the recrudescence rates are too high may be related to the lower exposure level during treatment with Artemotil. Therapy with Artemotil for severe malaria might be used in combination with a long-acting antimalarial such as mefloquine to avoid this problem with recrudescence.

The therapeutic efficacy was higher in the patients treated the high-dose regimens; a significant difference was found in the malaria cure rates (23% in low-dose patients and 48% in high-dose patients). The rate of recrudescence was higher in low-dose patients (77%) than in high-dose patients (46%). The pharmacokinetic data showed that the AUC of Artemotil in the high-dose group was 1.2-fold higher than that of the low-dose group. Similar results were also exhibited for the AUC value of DHA. This further indicated the high drug concentration (drug exposure level) produced a better therapeutic efficacy for Artemotil. Although the larger loading dose (high-dose regimen) for Artemotil gave higher AUC and Cmax values, it was also highly variable. This may be a particular problem in severe malaria, where peripheral blood flow may be reduced, and absorption from the depot sites may be further impeded. A better solution for reliable bioavailability would be an improved formulation with more rapid absorption.

Considerable interindividual variation in the pharmacokinetics of both parent drug and metabolite were reported. Dihydroartemisinin is the major plasma metabolite of Artemotil, which has been shown in vitro to be a five-fold more potent antimalarial than the parent drug.51,52 Thus, evaluating the conversion rate of Artemotil to DHA is very important for assessing the efficacy of Artemotil. In the present study, the two dose regimens showed a very low and similar conversion range (no significant difference) of Artemotil to DHA with 0.16–0.19 DHA to Artemotil ratios in rats. The low concentration and extended half-life of DHA did not contribute to the efficacy observed in this study, further suggesting that drug exposure level is more important than drug exposure time.

In conclusion, due to slow and prolonged absorption, Artemotil produced low peak concentrations, high accumulations, and longer exposure times in blood. Although Artemotil is therapeutically at least as effective as quinine for the treatment of cerebral malaria in children,4 the two dose regimens in this study failed to show a better therapeutic effect than that of other artemisinin drugs. In vitro and in vivo research has demonstrated that a high drug concentration in blood at first exposure is important for the extermination of the parasites, especially in conditions of severe malaria. The PCT seen in patient 151 shows the potential therapeutic effect of an intramuscular Artemotil, which is related to direct injection into a blood vessel with a rapid absorption and very high peak concentration. Therefore, a better solution for potential efficacy of this drug would be an improved formulation with more rapid and complete absorption.

Received February 5, 2004. Accepted for publication June 30, 2004.

Financial support: This work was supported by the UNDP/World Bank/World Health Organization Special Program for Research and Training in Tropical Diseases (TDR) and a Mahidol University Grant.

Authors’ addresses: Qigui Li, and Wilbur K. Milhous, Division of Experimental Therapeutics, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD 20910-7500, Telephone: 301-319-9351, Fax: 301-319-7360, E-mail: qigui.li{at}na.amedd.army.mil. Charles B. Lugt, ARTECEF BV, PO Box 5, NL-3600, AA Maarssen, The Netherlands. Sornchai Looareesuwan, Srivicha Krudsood, Polrat Wilairatana, Suparp Vannaphan, and Kobsiri Chalearmrult, Faculty of Tropical Medicine, Mahidol University, Hospital for Tropical Diseases, 420/6 Ratchavithi Road, 10400 Bangkok, Thailand, Telephone: 66-2-247-1688, Fax: 66-2-245-7288.

Reprint requests: Qigui Li, Division of Experimental Therapeutics, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD 20910-7500.


REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Davidson DE, 1994. Role of arteether in the treatment of malaria and plans for further development. Trans R Soc Trop Med Hyg 88 (Suppl 1): 51–52.
  2. Kager PA, Schultz MJ, Zijlstra EE, van Berg B, van Boxtel ChJ, 1994. Arteether administration in humans: preliminary studies of pharmacokinetics, safety and tolerance. Trans R Soc Trop Med Hyg 88 (Suppl 1): 53–54.[Web of Science][Medline]
  3. Looareesuwan S, Wilairatana P, 1998. The rational use of qinhaosu and its derivatives: what is the future of new compounds? Med Trop (Mars) 58 (Suppl 3): 89S–92S.
  4. Thuma PE, Bhat GJ, Mabeza GF, Osborne C, Biemba G, Shakankale GM, Peeters PAM, Oosterhuis B, Lugt CB, Gordeuk VR, 2000. A randomized controlled trial of artemotil (ß-arteether) in Zambian children with cerebral malaria. Am J Trop Med Hyg 62: 524–529.[Abstract]
  5. Moyou-Somo R, Tietche F, Ondoa M, Kouemeni LE, Ekoe T, Mbonda E, Nsangou C, Jemea B, Guemkam G, 2001. Clinical trial of beta-arteether versus quinine for the treatment of cerebral malaria in children in Yaounde, Cameroon. Am J Trop Med Hyg 64: 229–232.[Abstract]
  6. de Vries PJ, Dien TK, 1996. Clinical pharmacology and therapeutic potential of artemisinin and its derivatives in the treatment of malaria. Drugs 52: 818–836.[Web of Science][Medline]
  7. Looareesuwan S, Oosterhuis B, Schilizzi BM, Sollie FA, Wilairatana P, Krudsood S, Lugt CB, Peeters PA, Peggins JO, 2002. Dose-finding and efficacy study for i.m. artemotil (beta-arteether) and comparison with i.m. artemether in acute uncomplicated P. falciparum malaria. Br J Clin Pharmacol 53: 492–500.[Web of Science][Medline]
  8. Mishra SK, Asthana OP, Mohanty S, Patnaik JK, Das BS, Srivastava JS, Satpathy SK, Dash S, Rath PK, Varghese K, 1995. Effectiveness of {alpha}/ß-arteether in acute falciparum malaria. Trans R Soc Trop Med Hyg 89: 299–301.[Medline]
  9. Mohanty S, Mishra SK, Satpathy SK, Dash S, Patnaik J, 1997. Alpha, beta-arteether for the treatment of complicated falciparum malaria. Trans R Soc Trop Med Hyg 91: 328–330.[Medline]
  10. Mohapatr PK, Khan AM, Prakash A, Mahanta J, Srivastava VK, 1996. Effect of arteether alpha/beta on uncomplicated falciparum malaria cases in Upper Assam. Indian J Med Res 104: 284–287.[Medline]
  11. Singh N, Shukla MM, Asthana OP, Sharma VP, 1998. Effectiveness of alpha-beta arteether in clearing Plasmodium falciparum parasitemia in central India (Madhya Pradesh). Southeast Asian J Trop Med Public Health 29: 225–227.[Medline]
  12. Valecha N, Gupta S, Usha D, Biswas S, Sharma A, Adak T, Asthana OP, Sharma VP, 1997. Efficacy of alpha, beta-arteether in acute uncomplicated P. falciparum malaria. Int J Clin Pharmacol Res 17: 11–15.[Web of Science][Medline]
  13. van Hensbroek MB, van Onyiorah E, Jaffar S, Schneider G, Palmer A, Frenkel J, Enwere G, Forck S, Nusmeijer A, Bennett S, Greenwood B, Kwiatkowski D, 1996. A trial of arte-mether or quinine in children with cerebral malaria. N Engl J Med 335: 69–75.[Abstract/Free Full Text]
  14. Tran TH, Day NP, Nguyen HP, Nguyen TH, Tran TH, Pham PL, Dinh XS, Ly VC, Ha V, Waller D, Peto TE, White NJ, 1996. A controlled trial of artemether or quinine in Vietnamese adults with severe falciparum malaria. N Engl J Med 335: 76–83.[Abstract/Free Full Text]
  15. Karbwang J, Na-Bangchang K, Thanavibul A, Molunto P, 1998. Plasma concentrations of artemether and its major plasma metabolite, dihydroartemisinin, following a 5-day regimen of oral artemether, in patients with uncomplicated falciparum malaria. Ann Trop Med Parasitol 92: 31–36.[Medline]
  16. Looareesuwan S, Wilairatana P, Viravan C, Vanijanonta S, Pitisutitithum P, Kyle DE, 1997. Open randomized trial of oral artemether alone and a sequential combination with mefloquine for acute uncomplicated falciparum malaria. Am J Trop Med Hyg 56: 613–617.
  17. Mohamed SS, Khalid SA, Ward SA, Wan TS, Tang HP, Zheng M, Haynes RK, Edwards G, 1999. Simultaneous determination of artemether and its major metabolite dihydroartemisinin in plasma by gas chromatographymass spectrometry-selected ion monitoring. J Chromatogr B Biomed Sci Appl 731: 251–260.[Medline]
  18. Murphy S, English M, Waruiru C, Mwangi I, Amukoye E, Crawley J, Newton C, Winstanley P, Peshu N, Marsh K, 1996. An open randomized trial of artemether versus quinine in the treatment of cerebral malaria in African children. Trans R Soc Trop Med Hyg 90: 298–301.[Web of Science][Medline]
  19. Na-Bangchang K, Karbwang J, Thomas CG, Thanavibul A, Sukontason K, Ward SA, Edwards G, 1994. Pharmacokinetics of artemether after oral administration to health Thai males and patients with acute, uncomplicated falciparum malaria. Br J Clin Pharmacol 37: 249–253.[Medline]
  20. Salako LA, Walker O, Sowunmi A, Omokhodion SJ, Adio R, Oduola AM, 1994. Artemether in moderately severe and cerebral malaria in Nigerian children. Trans R Soc Trop Med Hyg 88 (Suppl 1): 13–15.
  21. Sowunmi A, Oduola AMJ, 1996. Efficacy of artemether in severe falciparum malaria in African children. Acta Trop 61: 57–63.[Web of Science][Medline]
  22. Walker O, Salako LA, Omokhodion SI, Sowunmi A, 1993. An open randomized comparative study of intramuscular artemether and intravenous quinine in cerebral malaria in children. Trans R Soc Trop Med Hyg 87: 564–566.[Web of Science][Medline]
  23. Barradell LB, Fitton A, 1996. Artesunate, A review of its pharmacology and therapeutic efficacy in the treatment of malaria. Drugs 50: 714–741.
  24. Li GQ, Wang XH, Guo XB, Fu LC, Jian HX, Chen PQ, 1999. Dose finding of dihydroartemisinin in treatment of falciparum malaria. Southeast Asian J Trop Med Public Health 30: 17–19.
  25. Looareesuwan S, Wilairatana P, Vanijanonta S, Pitisuttithum P, Viravan C, 1996. Treatment of acute, uncomplicated, falciparum malaria with oral dihydroartemisinin. Ann Trop Med Parasitol 90: 21–28.[Web of Science][Medline]
  26. Wilairatana P, Chanthavanich P, Singhasivanon P, Treeprasertsuk S, Krudsood S, Chalermrut K, Phisalaphong C, Kraisintu K, Looareesuwan S, 1998. A comparison of three different dihydroartemisinin formulations for the treatment of acute uncomplicated falciparum malaria in Thailand. Int J Parasitol 28: 1213–1218.[Medline]
  27. Xu C, Ding Y, Qi Z, 1997. Efficacy of dihydroartemisinin in treatment of 37 malaria cases. Chin J Intern Med 36: 187–189.
  28. Noedl H, Wernsdorfer WH, Krudsood S, Wilairatana P, Kollaritsch H, Wiedermann G, Looareesuwan S, 2001. Antimalarial activity of azithromycin, artemisinin and dihydroartemisinin in fresh isolates of Plasmodium falciparum in Thailand. Acta Trop 80: 39–44.[Medline]
  29. Shmuklarsky MJ, Klayman DL, Milhous WK, Rossan RN, Ager AL, Tang DB Jr, Heiffer MH, Canfield CJ, Schuster BG, 1993. Comparison of ß-artemether and ß-arteether against malaria parasites in vivo and in vitro. Am J Trop Med Hyg 48: 377–384.
  30. Bustos MDG, Gay F, Diquet B, 1994. In vitro tests on Philippine isolates of Plasmodium falciparum against four standard anti-malarials and four qinghaohu derivatives. Bull World Health Organ 72: 729–735.[Web of Science][Medline]
  31. Kager PA, 2003. Three newly registered drugs in the Netherlands for the treatment and chemoprophylaxis of malaria: atovaquone-proguanil, artemether-lumefantrine and Artemotil. Ned Tijdschr Geneeskd 147: 291–295.[Medline]
  32. Melendez V, Peggins JO, Brewer TG, Theoharides AD, 1991. Determination of the antimalarial arteether and its deethylated metabolite dihydroartemisinin in plasma by high-performance liquid chromatography with reductive electrochemical detection. J Pharm Sci 80: 132–138.[Medline]
  33. Li QG, Peggins JO, Fleckenstein L, Masonic K, Heiffer MH, Brewer TG, 1998. Bioavailability and pharmacokinetics of dihydroartemisinin, arteether, artemether, artesunic acid and artelinic acid in rats. J Pharm Pharmacol 50: 173–182.[Web of Science][Medline]
  34. Classen W, Altmann B, Gretener P, Souppart C, Skelton-Stroud P, Krinke G, 1999. Differental effects of orally versus parenterally administered qinghaosu derivatives artemether in dogs. Exp Toxicol Pathol 51: 507–516.[Web of Science][Medline]
  35. Li QG, Brueckner RP, Peggins JO, Brewer TG, 1999. Arteether pharmacokinetics in rats after 25 mg/kg/day single and multiple doses. Eur J Drug Metab Pharmacokinet 24: 283–294.
  36. Titulaer HAC, Zuidema J, Lugt CB, 1991. Formulation and pharmacokinetics of artemisinin and its derivatives. Int J Pharm 69: 83–92.
  37. Lugt CB, 2000. Dutch registration for artemotil injections. TDR News 63: 13.
  38. van Agtmael MA, Gupta V, van der Graaf CA, van Boxtel CJ. 1999. The effect of grapefruit juice on the time-dependent decline of artemether plasma levels in healthy subjects. Clin Pharmacol Ther 66: 408–414.[Web of Science][Medline]
  39. van Agtmael MA, Shan CQ, Jiao XQ, Mul R, van Boxtel CJ, 1999. Multiple dose pharmacokinetics of artemether in Chinese patients treated for falciparum malaria. Int J Antimicrob Agents 12: 151–158.[Web of Science][Medline]
  40. Ashton M, Nguyen DS, Nguyen VH, Gordi T, Trinh NH, Dinh XH, Nguyen TN, Le DC, 1998. Artemisinin kinetics and dynamics during oral and rectal treatment of uncomplicated malaria. Clin Pharmacol Ther 63: 482–493.[Web of Science][Medline]
  41. Ashton M, Hai TN, Sy ND, 1998. Artemisinin pharmacokinetics is time-dependent during repeated oral administration in healthy male adults. Drug Metab Dispos 26: 2601–2605.
  42. Khanh NX, de Vries PJ, Ha LD, van Boxtel CJ, Koopmans R, Kager PA, 1999. Declining concentrations of dihydroartemisinin in plasma during 5-day oral treatment with artesunate for falciparum malaria. Antimicrob Agents Chemother 43: 690– 692.[Abstract/Free Full Text]
  43. Park BK, O’Neill PN, Maggs JL, Pirmohamed M, 1998. Safety assessment of peroxide antimalarials: clinical and chemical perspectives. Br J Clin Pharmacol 46: 521–529.[Medline]
  44. Giao PT, de Vries PJ, 2001. Pharmacokinetic interactions of antimalarial agents. Clin Pharmacokinet 40: 343–373.[Medline]
  45. Alin MH, Bjorkman A, 1994. Concentration and time dependence of artemisinin efficacy agaist Plasmodium falciparum in vitro. Am J Trop Med Hyg 50: 771–776.
  46. Li QG, Mog SR, Si YZ, Kyle DE, Gettayacamin M, Milhous WK, 2002. Neurotoxicity and efficacy of arteether related to its exposure times and exposure levels in rodents. Am J Trop Med Hyg 66: 516–525.[Abstract]
  47. White NJ, Krishna S, 1989. Treatment of malaria: some considerations and limitations of the current methods of assessment. Trans R Soc Trop Med Hyg 83: 767–777.[Web of Science][Medline]
  48. White NJ, 1992. Antimalarial pharmacokinetics and treatment regimens. Br J Clin Pharmacol 34: 1–10.[Web of Science][Medline]
  49. World Health Organization, 2000. Severe falciparum malaria (Severe and Complicated Malaria, third edition). Trans R Soc Trop Med Hyg 94 (Suppl 1): 1–90.[Web of Science][Medline]
  50. Krudsood S, Wilairatana P, Vannaphan S, Treeprasertsuk S, Silachamroon U, Phomrattanaprapin W, Gourdeuk VR, Brittenham GM, Looareesuwan S, 2003. Clinical experience with intravenous quinine, intramuscular artemether and intravenous artesunate for the treatment of severe malaria in Thailand. Southeast Asian J Trop Med Public Health 34: 54–61.[Medline]
  51. Hassan AM, Bjorkman A, Ashton M, 1990. In vitro activity of artemisinin, its derivatives, and pyronaridine against different strains of Plasmodium falciparum. Trans R Soc Trop Med Hyg 84: 635–637.[Web of Science][Medline]
  52. Lee IS, Hufford CD, 1990. Metabolism of antimalarial sesquiterpene lactones. Pharmacol Ther 48: 345–355.[Web of Science][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by LI, Q.
Right arrow Articles by MILHOUS, W. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by LI, Q.
Right arrow Articles by MILHOUS, W. K.
Related Collections
Right arrow Malaria

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS