Introduction Plasmodium falciparum 1 2 3 5 −1 6 7 8 1 9 10 11 12 No pharmacokinetic interaction data have yet been published on amodiaquine when used in combination with artesunate. In view of the forthcoming widespread use of this combination in Africa, our study aimed to investigate potential pharmacokinetic interactions between these antimalarials and assess their tolerability in healthy volunteers in Africa. Methods Study subjects and study design Male or female healthy normal volunteers who provided written informed consent and met the following criteria were eligible to participate in the study: aged 18–45 years; no abnormalities on medical history, clinical examination, laboratory safety assessment (full blood count, differential white cell count, routine liver and renal function tests) or electrocardiogram; and a negative pregnancy test (for female volunteers). Volunteers were excluded if they were smokers (>5 cigarettes/day), had taken antimalarials or been in a malarial area in the preceding 8 weeks, had malaria parasites on a thick smear, used recreational drugs, or had ingested any alcohol or any medicines (including over-the-counter preparations) in the week preceding study commencement. This was a randomised three-phase crossover study. All volunteers took artesunate (4 mg/kg as Arsumax, 50 mg tablets, Sanofi-Aventis), the drug with the shorter elimination half-life, in the first phase, prior to the administration of amodiaquine. In the second phase, 7 days later, the volunteers were randomly allocated to one of two treatment groups: group 1 received a single oral dose of artesunate (4 mg/kg) plus amodiaquine (10 mg base/kg as Camoquin, 200 mg tablets, Parke Davis), and group 2 received a single oral dose of amodiaquine (10 mg/kg) alone. In phase three, 21 days later, the groups received the alternative regimen. All trial drugs were given under direct supervision with 200 ml tap water on an empty stomach after an overnight fast. No caffeinated drinks were allowed during the study period. Standardised meals were offered, commencing with breakfast at 2 h post-dose. Volunteers in phase one had blood samples collected before taking artesunate and at 0.25, 0.50, 0.75, 1.0, 1.5, 2, 3, 4, 6, 8 and 12 h after artesunate administration. In phases two and three (i.e. volunteers receiving artesunate plus amodiaquine or amodiaquine alone) venous blood samples were collected pre-dose and at 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 12, 24, 48 and 72 h, and days 4, 5, 7, 10, 14 and 20 post-dose. At each time two 5-ml venous blood samples were collected in tubes containing sodium heparin anticoagulant, while three 5-ml samples were collected at each time point for the first 12 h from those volunteers receiving both drugs. Each sample was centrifuged within 5 min of collection and the plasma transferred to separate plastic cryotubes and frozen at −70°C until analysed for artesunate, dihydroartemisinin, amodiaquine and desethylamodiaquine. Volunteers remained in the study ward for the first 12 h of each phase; thereafter specimens were collected at outpatient visits. 13 14 Ethical approval This clinical study was conducted in accordance with the principles laid down by the World Health Assembly of 1975 on Ethics in Human Experimentation and the Helsinki Declaration. The study adhered to the standards established for good clinical practice. The protocol was approved by the University of Cape Town Research Ethics Committee and the WHO Secretariat Committee for Research Involving Human Subjects (SCRIHS). Each volunteer was informed of the objectives, nature and possible risks of the trial. Written informed consent was obtained from every volunteer participating in the study. The volunteers were informed that they were free to withdraw consent at any time. Drug assays 15 Amodiaquine and desethylamodiaquine were analysed by LC mass spectrometry using an Agilent 1100 Series LC/MS system. Protein was precipitated from plasma samples (200 ul) using three volumes of acetonitrile. Supernatant (5 μl) was injected onto the HPLC. Chromatography was carried out using a 50 × 4.6 mm C18 Xterra column (Waters). The mobile phase comprised 75% acetonitrile, 0.02 M ammonia, pH 10.2. The extracted ion for amodiaquine was m/z 356 and for desethylamodiaquine m/z 328. The calibration curve for amodiaquine was linear in the range 5–100 ng/ml and for desethylamodiaquine in the range 5–400 ng/ml. Quality control samples of 25, 75 and 250 ng/ml were used for desethylamodiaquine and 2.5, 7.5 and 25 ng/ml for amodiaquine. Within- and between-day coefficients of variation were below 12%. The lower limit of quantification was 5 ng/ml for both amodiaquine and desethylamodiaquine. Statistical analysis d 1/2 d 1/2 d max 16 17 9 10 18 The pharmacokinetic parameters following administration of artesunate and amodiaquine alone and in combination were compared using Stata, version 9.0 [Stata, College Station, TX, USA]. Data were log-transformed and then compared using the analysis of variance (ANOVA) for a cross-over design to take into account the repeated measures by study subject, treatment period and treatment groups and, for amodiaquine and desethylamodiaquine, a sequence effect. The treatment effects generated from the ANOVA were exponentiated in order to express comparisons between monotherapy and combination therapy as a ratio. Any apparent discrepancies between the difference in the group means and these ratios are due to the fact that the ratios are based on the within-patient differences in log-transformed values and not the group arithmetic means. Given the multiple testing, statistical significance of results should be interpreted with caution. For the safety analysis, means and 95% confidence intervals (95% CI) for each haematological parameter (haemoglobin, haematocrit, platelets, white cell count, absolute lymphocyte and neutrophil count) were calculated at baseline and at the end of each treatment phase. Changes in these values from baseline were determined using a mixed effect regression model that took into account repeated measures within-patient and were adjusted for the period and period-treatment interaction effects. Results Subject demographics Nineteen volunteers were screened; two were excluded because of neutropaenia and two withdrew consent. Fifteen healthy normal volunteers (10 male, 5 female) entered the study with a mean age, weight and height of 24.4 years, 67.3 kg and 171 cm, respectively. The mean dose of amodiaquine was 10.72 mg/kg and that of artesunate was 4.26 mg/kg. Safety analysis included all 15 volunteers; only data from the 13 volunteers who completed the study were included in the pharmacokinetic analyses. A female volunteer was withdrawn due to a new prescription of fluoxetine for depression, as this drug could potentially interact with artesunate and amodiaquine by inhibiting cytochrome P450 enzymes. One male volunteer was withdrawn due to a possible hypersensitivity reaction on his first exposure to amodiaquine. Pharmacokinetic parameters Effect of amodiaquine on artesunate pharmacokinetic parameters 1 max max max 1 2 P max P 1/2 P d P 1 max max P P max 1 Fig. 1 mono combo a AS DHA b AQ DEAQ  Table 1 Pharmacokinetic parameters for artesunate and dihydroartemisinin when artesunate was administered alone and with amodiaquine (ACT)   AUC (ng·h/ml) max max 1/2 d Cl/f (l/min) Artesunate AS alone (mean ± SD) 206.4 ± 135.5 231.8 ± 155.0 0.62 ± 0.28 NA NA NA ACT (mean ± SD) 183.3 ± 146.5 141.6 ± 117.5 0.86 ± 0.67 NA NA NA ACT-to-monotherapy ratio (%) (mean, 95% CI) 36 (8–173) 25 (6–105) 125 (76–202) NA NA NA   Significance (ANOVA) 0.18 0.057 0.33    Dihydroartemisinin AS alone (mean ± SD) 2,044.4 ± 564.2 844.5 ± 309.4 1.10 ± 0.95 1.46 ± 0.48 4.89 ± 1.67 2.46 ± 0.86 ACT (mean ± SD) 1,410.5 ± 543.6 446.2 ± 239.5 2.08 ± 1.72 2.20 ± 0.85 9.68 ± 4.16 3.08 ± 0.82 ACT-to-monotherapy ratio (%) (mean, 95% CI) 67 (51–88) 51 (33–78) 165 (82–334) 157 (115–213) 192 (133–275) 122 (96–156)   Significance (ANOVA) 0.008 0.005 0.15 0.008 0.003 0.097 Treatment effects generated from the ANOVA were exponentiated to express within-subject comparisons between monotherapy and combination therapy as a ratio, adjusted for period effects C max T max max T 1/2 V d f Cl AS SD CI NA 1/2 d Fig. 2 a b  Effect of artesunate on amodiaquine pharmacokinetic parameters 1 2 2 3 Fig. 3 Stick plot comparing individual patients. Day 7 concentrations of desethylamodiaquine when amodiaquine was administered as monotherapy and in combination with artesunate. (Note: volunteer A was excluded from the statistical analysis)  2 P max P P 2 P 3 d max Table 2 Pharmacokinetic parameters for desethylamodiaquine (DEAQ) when amodiaquine was administered alone and with artesunate (ACT)   AUC (ng·h/ml) max max [Day 7] (ng/ml) 1/2 d Cl/f (l/min) Amodiaquine AQ alone (mean ± SD) 162.4 ± 101.4 29.2 ± 10.9 2.32 ± 1.16 NA 5.3 ± 4.1 361.0 ± 128.3 69 ± 59 ACT (mean ± SD) 108.5 ± 56.0 22.7 ± 9.0 2.18 ± 1.61 NA 3.9 ± 1.2 467.7 ± 180.5 86 ± 26 ACT-to-monotherapy (%) (mean, 95% CI) 77 (47–127) 78 (58–103) 92 (57–147) NA 74 (29–189) 128 (93–175) 172 (84–357)   Significance (ANOVA) 0.27 0.07 0.68  0.44 0.11 0.11 Desethylamodiaquine AQ alone (mean ± SD) 12,041 ± 3,480 268.7 ± 70.8 3.68 ± 1.85 19.4 ± 7.3 240.8 ± 146.9 234.1 ± 97.5 768 ± 252 ACT (mean ± SD) 8,437 ± 4,009 301.4 ± 166.1 2.18 ± 1.03 13.3 ± 7.3 136.9 ± 83.8 210.8 ± 92.9 1,330 ± 735 ACT-to-monotherapy ratio (%) (mean, 95% CI)) 65 (46–90) 103 (73–147) 60 (45–80) 56 (30–104) 53 (25–111) 88 (51–149) 164 (112–243)   Significance (ANOVA) 0.015 0.82 0.003 0.064 0.08 0.58 0.016 Treatment effects generated from the ANOVA have been exponentiated to express within-subject comparisons between monotherapy and combination therapy as a ratio, adjusted for period and sequence effects AUC C max T max max [Day 7] T 1/2 V d f Cl AQ SD CI NA Safety P 7 9 9 9 There were no significant changes following treatment seen on the ECGs; the mean (95% CI) QTc interval was 398 (390–406) ms at screening, 401 (393–409) ms following artesunate alone, 400 (385–414) ms following amodiaquine alone and 412 (400–424) ms following the combination. In two patients, the prolongation following treatment with amodiaquine or artesunate plus amodiaquine was considered of borderline clinical significance. Discussion The pharmacokinetic analyses in this randomised, cross-over study showed statistically significant pharmacokinetic interactions resulting in reductions in the AUC of both dihydroartemisinin and desethylamodiaquine when artesunate and amodiaquine were given in combination to healthy volunteers. Of further concern is that one healthy volunteer failed to reach quantifiable concentration of both artesunate and dihydroartemisinin throughout the initial 12 h. There is insufficient evidence currently available to explain the basis of these interactions. max 0–12 d max 19 20 21 22 23 23 18 24 1 6 max 1 6 1 To conclude, the total exposure to the main active metabolites of both artesunate and amodiaquine was significantly reduced when administered in combination to healthy African volunteers; this might be important clinically. However, because cure rates with this combination are generally higher than with amodiaquine monotherapy, artesunate and amodiaquine could remain in the armamentarium of drugs used to combat falciparum malaria, provided efficacy continues to be monitored and adequate safety precautions can be taken. Further pharmacokinetic research on artesunate plus amodiaquine, when administered concurrently or as a fixed dose combination, is urgently required in patients with malaria to establish the extent and clinical significance of these pharmacokinetic interactions.