Pharmacokinetics and Ex Vivo Pharmacodynamic Antimalarial Activity of Dihydroartemisinin-Piperaquine in Patients with Uncomplicated Falciparum Malaria in Vietnam

Compared to healthy subjects, malaria patients show a reduction in the mean oral clearance (1.19 versus 5.87 liters/h/kg of body weight) and apparent volume of distribution (1.47 versus 8.02 liters/kg) of dihydroartemisinin in Vietnamese patients following treatment with dihydroartemisinin-piperaquine (Artekin) for uncomplicated Plasmodium falciparum. Dihydroartemisinin is responsible for most of the ex vivo antimalarial activity of dihydroartemisinin-piperaquine.Dihydroartemisinin-piperaquine (Artekin) is an artemisinin-based combination treatment (ACT) drug that is well tolerated and highly effective in the treatment of Plasmodium falciparum malaria in Southeast Asia and Africa, with cure rates typically greater than 95% following a standard 3-day course (8, 9, 15, 17). Despite its extensive use over the past 5 years, no data are available on the clinical pharmacokinetics of dihydroartemisinin in malaria patients following treatment with the ACT. Few studies have investigated the pharmacokinetics of piperaquine in malaria patients. The highly lipophilic drug exhibits biphasic disposition kinetics, with a large apparent volume of distribution, low oral clearance, and a lengthy elimination half-life of about 3 to 4 weeks in malaria patients treated with dihydroartemisinin-piperaquine (6, 18).The present study investigated the clinical pharmacokinetic properties of dihydroartemisinin and piperaquine after a 3-day course of dihydroartemisinin-piperaquine in the treatment of uncomplicated P. falciparum malaria in Vietnamese patients. In addition to assessing the in vivo response of the ACT, the ex vivo pharmacodynamic antimalarial activity of dihydroartemisinin-piperaquine in the patients'' plasma samples was investigated against two lines of P. falciparum. The study was conducted at Military Hospital 175 in Ho Chi Minh City, Vietnam, in 12 adult Vietnamese patients. The volunteers had to satisfy the following inclusion criteria: male, aged 17 to 55 years old, parasite density between 500 and 100,000 per μl of blood, axillary temperature of ≥37.5°C or a history of fever in the previous 24 h, written informed consent, and willing to be monitored for 35 days. The exclusion criteria were as follows: antimalarial treatment within the preceding 2 weeks, mixed plasmodial infection, and history of another serious medical disease. The patients acquired their infections in Binh Phuoc Province, about 120 km from Ho Chi Minh City. The patients stayed at the hospital for the entire 35-day follow-up period and because Ho Chi Minh City is free of malaria, the possibility of reinfection was avoided. Ethical approval for the study was obtained from the Review and Scientific Board of Military Hospital 175 and the Australian Defense Human Research Ethics Committee (ADHREC protocol 379/05).The patients were administered a weight-based 3-day course of dihydroartemisinin-piperaquine (Holleykin Pharmaceuticals, China) (each tablet contained 40 mg of dihydroartemisinin and 320 mg of piperaquine phosphate) at 2.4 mg of dihydroartemisinin per kg of body weight and 19.2 mg of piperaquine per kg of body weight per day, rounded up or down to the nearest half tablet, with day 0 being designated the first dose. Dihydroartemisinin-piperaquine was administered within 15 min of having a standard Vietnamese breakfast of rice, noodles, and meat to enhance the absorption of piperaquine (13). Parasitemia and axillary temperature were measured before commencement of treatment and then every 8 h afterwards to determine parasite and fever clearance times. Blood samples (7 ml) were collected at 0, 0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 10, and 12 h using an indwelling cannula kept patent with heparinized saline after the last dose of dihydroartemisinin-piperaquine on day 2. Subsequent heparinized blood samples were collected by venipuncture at days 3, 4, 7, 14, 21, 28, and 35 after commencement of treatment. Blood samples were centrifuged, and the separated plasma samples were stored at −25°C until transported on dry ice to Australia for drug analysis, which was within 12 months of collection.Plasma dihydroartemisinin concentrations were measured by liquid chromatography-tandem mass spectrometry, with a lower limit of quantification of 1 ng/ml (2). The values for overall precision of analysis for dihydroartemisinin, as defined by the percent coefficient of variation of spiked samples were 6.3% at 1 ng/ml, 5.7% at 20 ng/ml, 4.6% at 200 ng/ml, and 6.6% at 750 ng/ml. The corresponding inaccuracy values were 1.3%, 1.5%, 0.7%, and 3.2% for 1, 20, 200, and 750 ng/ml, respectively. Plasma piperaquine concentrations were measured by a validated high-performance liquid chromatography method, with a lower limit of quantification of 5 ng/ml (11). The precision values of the assay were 10.3% at 10 ng/ml, 6.8% at 100 ng/ml, 6.7% at 500 ng/ml, and 6.5% at 1,000 ng/ml. The corresponding inaccuracy values were 11.1%, 2.8%, 0.8%, and 0.5% for 10, 100, 500, and 1,000 ng/ml, respectively. Pharmacokinetic parameters (peak concentration [C_max], time to reach maximum concentration [T_max], area under the concentration-time curve from day 2 to the last data point [AUC_d2-ld] and from day 2 to infinity [AUC_d2-∞], terminal half-life [t_1/2], apparent oral clearance, and apparent volume of distribution) were determined from the plasma concentration-time data using noncompartmental methods.The ex vivo pharmacodynamic antimalarial activity of dihydroartemisinin-piperaquine was assessed by culturing malaria parasites in vitro in the presence of patients'' plasma samples collected after the last administration of dihydroartemisinin-piperaquine by the method of Kotecka et al. (10), with minor modifications. Briefly, patients'' plasma samples (50 μl) were serially diluted twofold on microtiter plates with drug-free human plasma. The in vitro drug susceptibility of two lines of P. falciparum (chloroquine-sensitive D6 and chloroquine-resistant K1) to dihydroartemisinin and piperaquine were determined in parallel with the patients'' plasma samples. Fifty microliters of spiked drug solutions prepared in human plasma were serially diluted twofold on microtiter plates using drug-free plasma. Inoculum (50 μl) was added to each well containing either the patient''s plasma samples or spiked drug solutions, so that the total cell suspension (100 μl) contained 50% plasma in hypoxanthine-free plain LPLF-RPMI 1640, with a final hematocrit of 2% and a parasitemia (>95% rings) of 1%. Tritiated hypoxanthine incorporation was used to determine the extents to which parasite growth was inhibited by different drug concentrations or dilutions of the patients'' plasma during 48 h of incubation. The inhibitory concentration (90% infective concentration [IC₉₀] for spiked drug samples) and inhibitory dilution (90% infective dose [ID₉₀] for patient plasma samples) were defined as the drug concentrations and the number of dilutions of the patient plasma sample, respectively, that produced a 90% inhibition of uptake of tritiated hypoxanthine by intraerythrocytic malaria parasites compared to drug-free plasma samples (controls).The mean (standard deviation) age of the patients was 26.3 (10.4) years, with a mean (standard deviation) weight of 56.5 (7.8) kg. The patients had an admission geometric mean parasitemia of 15,198 parasites/μl of blood (range, 738 to 79,310 parasites/μl) and a mean temperature (standard deviation) of 37.7°C (1.3°C), with 42% (5 of 12) of patients with fever. Treatment with dihydroartemisinin-piperaquine promptly reduced fever, with a median fever clearance time of 24 h (range, 16 to 48 h) and led to a rapid reduction in parasite density, with a median parasite clearance time of 28 h (range, 16 to 56 h). Over the 35-day follow-up period, there was no recurrence of infection in the patients. The mean content values of five dihydroartemisinin-piperaquine tablets were 105.4% ± 4.8% for dihydroartemisinin and 109.0% ± 1.1% for piperaquine. Although none of the patients reported treatment with antimalarial drugs 2 weeks before commencing dihydroartemisinin-piperaquine, no dihydroartemisinin or piperaquine was detected in their predose samples, which confirmed no recent treatment with either artesunate or CV8 (dihydroartemisinin-piperaquine-primaquine-trimethoprim).The mean plasma concentration-time profiles of dihydroartemisinin and piperaquine after a 3-day course of dihydroartemisinin-piperaquine are shown in Fig. ​Fig.1,1, and the pharmacokinetics of the two drugs are summarized in Table ​Table1.1. Because dihydroartemisinin is rapidly eliminated with a t_1/2 of about 1 h in healthy Vietnamese subjects (2) and does not accumulate with daily administration, we were able to compare the pharmacokinetics of dihydroartemisinin in the Vietnamese patients after the last daily dose of the 3-day course of dihydroartemisinin-piperaquine with values obtained in healthy Vietnamese subjects given a single dose of dihydroartemisinin-piperaquine (2). The mean C_max and AUC of dihydroartemisinin were markedly higher in the Vietnamese malaria patients than in the healthy Vietnamese subjects (C_max, 698 versus 176 ng/ml; AUC_d2-∞, 1,949 versus 398 ng·h/ml). Although the difference was not as large, Binh et al. (1) reported an approximately twofold-higher mean C_max (1,045 versus 480 ng/ml) and AUC (2,401 versus 932 ng·h/ml) of dihydroartemisinin in Vietnamese malaria patients compared with healthy subjects administered a single dose of dihydroartemisinin (120 mg) alone. Dihydroartemisinin was rapidly eliminated in the malaria patients with a t_1/2 of 0.85 ± 0.15 h. The apparent oral clearance and apparent volume of distribution of dihydroartemisinin were 4.9-fold lower (1.19 versus 5.87 liters/h/kg) and 5.5-fold lower (1.47 versus 8.02 liters/kg) in the Vietnamese malaria patients than in the healthy subjects, respectively (2). A reduction in clearance and contraction in the apparent volume of distribution has also been reported for other antimalarial drugs, such as mefloquine (7) and quinine (21), during the acute phase of malaria. A likely explanation for the increase in bioavailability of dihydroartemisinin in malaria patients compared with healthy subjects is a decrease in hepatic clearance of dihydroartemisinin due to malaria (1). Alpha-acid glycoprotein levels also increase during acute malaria (16), and similar to quinine, this may cause an increase in binding of the protein-bound dihydroartemisinin, with a reduction in the apparent volume of distribution of the drug (12).Open in a separate windowFIG. 1.Plasma dihydroartemisinin (○) and piperaquine (•) concentration-time profiles after the last dose of a 3-day course of dihydroartemisinin-piperaquine (2.4 mg of dihydroartemisinin per kg and 19.2 mg of piperaquine per kg daily) for the treatment of uncomplicated Plasmodium falciparum malaria in 12 Vietnamese patients. The values shown are means plus standard deviations (error bars). The inset shows the piperaquine concentrations from day 2 to day 35 after commencement of treatment. The ex vivo pharmacodynamic antimalarial activity profile (mean ID₉₀ values ▴) of patients'' plasma samples after dihydroartemisinin-piperaquine treatment is derived from the K1 line of P. falciparum.

TABLE 1.

Pharmacokinetic parameters of dihydroartemisinin and piperaquine in 12 Vietnamese patients with uncomplicated P. falciparum malaria^a

Drug	Pharmacokinetic parameter (mean ± SD)
	Cmax (ng/ml)	Tmax (h)	AUCd2-ld (ng·h/ml)	AUCd2-∞ (ng·h/ml)	Extrap. AUC (%)b	t1/2 (h)	CL/F (liters/h/kg)c	V (liters/kg)d
Dihydroartemisinin	698 ± 169	2.8 ± 1.1	1,946 ± 445	1,949 ± 445	0.14 ± 0.17	0.85 ± 0.15	1.19 ± 0.28	1.47 ± 0.46
Piperaquine	568 ± 288	5.7 ± 1.9	44,430 ± 17,435	56,418 ± 20,144	29.0 ± 22.1	427 ± 128

Open in a separate window^aPharmacokinetic parameters of dihydroartemisinin and piperaquine in patients with uncomplicated malaria after the last dose of a 3-day course of dihydroartemisinin-piperaquine (2.4 mg of dihydroartemisinin and 19.2 mg of piperaquine daily).^bExtrapolated (Extrap.) AUC (%) = [(AUC_d2-∞ − AUC_d2-ld)/(AUC_d2-∞)] × 100.^cCL/F, apparent oral clearance.^dV, apparent volume of distribution.In contrast to dihydroartemisinin, no data are available on the pharmacokinetics of piperaquine given as a 3-day course of dihydroartemisinin-piperaquine in healthy volunteers, and thus a comparison between malaria patients and healthy subjects could not be made to elucidate whether malaria affects the disposition of piperaquine after dihydroartemisinin-piperaquine administration. In the present study, the mean plasma concentration of piperaquine immediately before the last dose of dihydroartemisinin-piperaquine was 131 ng/ml (range, 48 to 261 ng/ml). The mean C_max of piperaquine was 568 ng/ml, which was reached 5.7 h after the final dose. The mean t_1/2 of piperaquine of 17.8 days in the Vietnamese malaria patients was less than 23 days in Cambodian adult malaria patients (6) and 28 days in Burmese and Thai Karen malaria patients (18). However, the estimated elimination half-life in the present study might have been underestimated, since piperaquine exhibits multiphasic elimination (19), with blood sampling limited to 35 days after starting dihydroartemisinin-piperaquine treatment. It has been previously reported that the day 7 piperaquine concentration is an important determinant of therapeutic response to dihydroartemisinin-piperaquine and that malaria patients with levels below 30 ng/ml are more likely to have a recurrence of malaria (14). All the malaria patients had day 7 piperaquine concentrations greater than 30 ng/ml, with a range of 37 to 118 ng/ml.In vitro drug susceptibility testing revealed that dihydroartemisinin was 15-times [mean IC₉₀ of 4.50 ± 0.25 versus 67.51 ± 1.87 nM (n = 3)] and 25.2-times [IC₉₀ of 4.85 ± 1.51 versus 122.16 ± 23.69 nM (n = 7)] more active than piperaquine in inhibiting the D6 and K1 lines of P. falciparum, respectively. The IC₅₀s for dihydroartemisinin (2.57 ± 1.27 nM) and piperaquine (82.06 ± 35.25 nM) were about twofold higher than previously published data using the K1 line (3, 4). A likely explanation for this discordance in IC₅₀s is the higher human plasma concentration (50% versus 10%) used in the present study compared with others using standard in vitro methods (M. Chavchich, unpublished data).The ex vivo pharmacodynamic antimalarial activity profile (ID₉₀ values) of dihydroartemisinin-piperaquine corresponded with the plasma concentration-time data of dihydroartemisinin from 0.5 h to 10 h after dosing (Fig. ​(Fig.1).1). Dihydroartemisinin''s superior potency, rapid onset of action, and broader blood-stage specificity (5, 20) compared to piperaquine appears to provide the major contribution to the rapid clearance of parasites and fever in the malaria patients. At the T_max of dihydroartemisinin, the mean numbers of dilutions of patients'' plasma samples required to produce an ID₉₀ were 639 and 513 against the D6 and K1 lines, respectively. However, by 12 h after the last dose of dihydroartemisinin-piperaquine, most of the antimalarial activity in the patients'' plasma samples was due solely to piperaquine. At day 7 after commencement of treatment, the mean number of dilutions of patients'' plasma samples required to produce an ID₉₀ had declined to <2 for both D6 and K1 lines. At day 28, the patients'' piperaquine concentrations (mean, 25 ng/ml; range, 10 to 42 ng/ml) were insufficient to completely kill either the D6 or K1 parasites. The clinical significance of the ex vivo pharmacodynamic antimalarial activity profile of dihydroartemisinin-piperaquine is that any mutant parasite that might survive physiological dihydroartemisinin and piperaquine concentrations during the 3-day treatment period of dihydroartemisinin-piperaquine will have to engage only the less active piperaquine. Furthermore, because of piperaquine''s prolonged terminal half-life and diminishing parasiticidal concentrations, selection pressure will be of concern for the potential emergence of resistant parasites to piperaquine.In conclusion, malaria infection affects the disposition of dihydroartemisinin, with a reduction in both the apparent volume of distribution and apparent oral clearance of the drug. During the first 10 h after dihydroartemisinin-piperaquine administration, the highly active and rapidly eliminated dihydroartemisinin contributes most of the ex vivo pharmacodynamic antimalarial activity of the dihydroartemisinin-piperaquine combination. After this period, the less active and slowly eliminated piperaquine will lead to selection pressure for the potential development of drug resistance, which may limit the future effectiveness of dihydroartemisinin-piperaquine, particularly in nonimmune malaria patients.