All three hybrids displayed potent low nanomolar in vitro anti-malarial activities, with IC50 values very closely related, ranging from 5.15 – 25.7 nM, whereas 2.09 – 5.11 nM and 21.54 – 157.90 nM were the ranges of DHA and CQ, respectively. The best in vitro activity against the 3D7 strain was displayed by hybrid 3 (IC50 = 5.15 nM). It was clear from the data presented that this class of compounds displayed a very potent and rapid in vivo anti-malarial activity when optimum dosages were applied, resulting in recrudescence if otherwise applied.
Hybrid 1 demonstrated a potent anti-malarial activity via both intraperitoneal and oral routes. At 15 mg/kg ip and 50 mg/kg per os rapid and total parasitaemia clearance were induced. No visible sign of toxicity was observed up to 50 mg/kg. No parasitaemia were observed on the smears form day 3. Long term monitoring showed 100% survival on day 30 and no recrudescence was observed. Artemisinin hybrid 1 provided a total cure of malaria in vivo at these doses.
Hybrid 2 provided a complete clearance of parasitaemia and a total cure of malaria at 15 mg/kg by ip route and at 50 mg/kg by oral route. Compared to hybrid 1, hybrid 2 displayed similar efficiency against P. vinckei in vivo at the same doses allowing a total cure and similar ED50 values (1.1 and 1.4 mg/kg ip for hybrid 1 and hybrid 2, respectively and 12 mg/kg and 16 mg/kg per os for hybrid 1 and hybrid 2, respectively).
Although the four day treatment with hybrid 3 provided good anti-malarial efficacy against P. vinckei in vivo at 2.5 mg/kg ip and 25 mg/kg per os (doses where parasitaemia is less than 10% on day 5), survival rate was 66%. Therefore, a complete clearance of parasitaemia and a total cure of malaria in vivo were not obtained by hybrid 3.
Artesunate, the reference drug, displayed ED50 ip and per os values of < 1 mg/kg and 1.8 mg/kg, respectively. However, despite these low ED50 values, artesunate was only able to completely cure mice at doses about 40 times higher. Clearance of parasitaemia was only obtained at 30 mg/kg ip and 80 mg/kg per os, whereas complete clearance was obtained at 15 mg/kg ip and 50 mg/kg per os for hybrid 1 and 2.
The pharmacokinetic profile (mean ± SD) of hybrid 2 was similar to that of DHA. Hybrid 2 reached a maximum concentration of 141 ± 56.8 ng/mL within 23 ± 5.77 min after a concentration of 20 mg/kg were administrated orally, whereas DHA reached a maximum concentration of 142.2 ± 21.1 ng/mL, in 48 ± 6 min after a dose of 10 mg/kg . However, according to the recorded data DHA displayed a much longer half-life ip than hybrid 2 (25 min vs 3.9 min) . Subsequently DHA also displayed a higher AUC than hybrid 2 (8748 ± 2016 ng.min/mL vs 3463 ± 895 ng.min/mL) . The oral volume of distribution of DHA was higher than that of hybrid 2 (353.5 ± 194.9 L/kg vs 34 ± 10.8 L/kg), whereas the clearance rate for hybrid 2 was 5 times that of DHA (6.1 L/min/kg vs 1.19 L/min/kg) . Hybrid 2 resulted in a significantly lower oral bioavailability, 0.31%, compared to 19 – 35%, which has been reported for artemisinin derivatives .
The high values for oral artemisinin clearance either indicated moderate absorption or high first-pass extraction, which also explain the time dependency of DHA  and hybrid 2. Moderate bioavailability displayed by hybrid 2 could be explained by rapid metabolism. However, metabolites – not identified by the LC/MS/MS assay – are expected to be very active due to their potent in vivo anti-malarial activity.