In Vztro Activity of Dihydroartemisinin against Clinical, Isolates of Plasmodzum Falczparum in Yaounde, Cameroon

The in vitro activities of dihydroartemkinin (the biologically active metabolite of artemisinin derivatives), chloroquine, monodesethylamodiaquine (the biologically active metabolite of amodiaquine), quinine, mefloquine, halofantrine, and pyrimethamine were assessed in 65 African isolates of Plasmodium falciparum from Yaounde, Cameroon using an isotopic microtest. The 50% inhibitory concentration (IC5,) values for dihydroartemisinin were within a narrow range from 0.25 to 4.56 nM, with a geometric mean of 1.1 1 nM (95% confidence interval = 0.96-1.28 nM). Dihydroartemisinin was equally active (P > 0.05) against the chloroquine-sensitive isolates (geometric mean IC,, = 1.25 nM, 95% confidence interval = 0.99-1.57 nM) and the chloroquine-resist isolates (geometric mean ICso = 0.979 IN, 95% confidence interval = 0.816-1.18 nM). A significant positive correlation was observed between the responses to dihydroartemisinin and mefloquine (r = 0.662) or halofantrine (r = 0.284), suggesting in vitro crossresistance. There was no correlation between the responses to dihydroartemisinin and other antimalaid drugs. Artemisinin (qinghaosu) has been used for centuries in traditional Chinese medicine for antimalarial treatment.’ In 1972, Chinese scientists isolated the active principle from the plant Artemisia annua.2 Artemisinin is a sesquiterpene lactone characterized by the presence of an endoperoxide that is associated with a potent antimalarial activity. Because artemisinin is chemically unstable and poorly soluble in water or oil, the carbonyl group at C-10 of the parent compound was reduced to obtain dihydroartemisinin. Several derivatives have been developed by adding an ether, ester, or other substituents to the hydroxyl gfDup of dihydroartemisinin. These semi-synthetic derivatives include the water-soluble derivatives, sodium artesunate and artelinic acid, and the oilsoluble derivatives, artemether and arteether. With the exception of arteether and artelinic acid, these compounds are used to treat malarial infections in endemic countries, mainly in Southeast Asia and, to a lesser extent, in Africa and South America. Artemisinin derivatives are available in different fomulations for oral, parenteral, and rectal administration. Clinical studies have shown that artemisinin derivatives are highly potent, rapidly acting, and well-tolerated blood schizontocides, resulting in shorter parasite clearance times than other antimalarial Art$misinin derivatives are highly effective against$Pl&modium falciparum isolates that are resistant to other drugs. These derivatives are indicated for the emergency treatment of severe and complicated falciparum malaria by parenteral administration and for the oral treatment of uncomplicated multidrug-resistant malaria? So far, resistance to artemisinin derivatives has not been reported in clinical studies. However, because of the high recrudescence rate when used as monotherapy, many current therapeutic regimens used in Southeast Asia include a combination of one of the artemisinin derivatives and mefloquine.&P The combination therapy with other drugs (lumefantrine, tetra. cycline, pyrimethamine, desferrioxamine) is under evaluaAlthough pharmacokinetic data are incomplete, it has been established that a l l currently available artemisinin derivatives are metabolized rapidly to dihydroartemisinin, the biologicdiy active máin-hum’än metabolite.’G17 In the case tion.10-13 of sodium artesunate, the aqueous solution that is reconstituted by dissolving the anhydrous powder of the drug in 5% dextrose solution just before parenteral administration is known to hydrolyze the compound rapidly into dihydroartemisinin.’* Initial in vitro studies on laboratory-adapted clones of P. falciparum have shown that dihydroartemisinin is more active than the other artemisinin de ri vat ive^.'^ Based on the+,observation that dihydroartemisinin is a highly active metabolite of artemisinin derivatives and that it is well absorbed by oral administration, Chinese investigators have developed oral dihydroartemisinin for clinical use. Dihydroartemisinin is not commercially available outside China. Results of the recent clinical study in Thailand showed the efficacy of oral dihydroartemisinin, with a cure rate of 90% in 49 patients treated for 7 days.20 Despite the absence of official therapeutic guidelines for the use of these derivatives, artemether and artesunate have been commercially available in Cameroon since 1997. We have determined the in vitro activity of artemether and artesunate in our previous studies.2’,22 In the present study, we assessed the in vitro response of fresh clinical isolates to dihydroartemisinin to 1) establish the baseline sensitivity level of dihydroartemisinin in Cameroon, 2) compare its in vitro activity with that of other standard antimalarial drugs, and 3) evaluate the in vitro cross-resistance pattems. MATERIALS AND METHODS Parasites. Sixty-five clinical isolates of P. falciparum were obtained before treatment from symptomatic, African patients attending the Nlongkak dispensary in Yaoundé between 1997 and 1998. The patients were screened for selfmedication by the urine test of Saker-Solomons, which was developed for the detection of chloroquine but also detects other 4-aminoquinolines, aminoalcohols, and antifolate drugs due to cross-reaction.u Giemsa-stained thin blood smears were examined for Plasmodium species identification, and parasite density was determined against 5,000 erythrocytes. Venous blood (10 ml) was collected in a tube coated with EDTA (Terumo Europe N. V., Leuven, Belgium) after patients’ informed consent was obtained. Blood sam-