Inhibitory effects of 9-anilinoacridines on Plasmodium falciparum gametocytes.

Two gametocyte-producing isolates of Plasmodium falciparum, KT1 arid KT3, were cultivated in vitro. On day 11 of cultivation, pure gametocytes containing stage II, III and IV were used to test the gametocytocidal activity of 9-anilinoacridines that had previously demonstrated their activity against the asexual stage of the parasite. After drug exposure for 48 h, gametocytes were maintained without drugs for another 2 days before thin films were prepared for parasite counting. Gametocytocidal activities of 13 analogs of 9-anilinoacridine were observed with 50% inhibitory concentrations in the range of 0.6 microM to> 100 microM The most active compound was 1'-CH2NMe2-9-anilinoacridine. Anilinoacridine derivatives with 3,6-diamino substitution had reduced gametocytocidal activity in contrast to their enhancing effect against the asexual forms. Morphological abnormalities of gametocytes were observed following drug exposure.

Gametocytocidal activity of pyronaridine and DNA topoisomerase II inhibitors against multidrug-resistant Plasmodium falciparum in vitro.

Gametocytocidal activities of pyronaridine and DNA topoisomerase II inhibitors against two isolates of multidrug-resistant Plasmodium falciparum, KT1 and KT3 were determined. After sorbitol treatment, pure gametocyte cultures of Plasmodium falciparum containing mostly young gametocytes (stage II and III) obtained on day 11 were exposed to the drugs for 48 h. The effect of the drugs on gametocyte development was assessed by counting gametocytes on day 15 of culture. Pyronaridine was the most effective gametocytocidal drug against P. falciparum isolates KT1 and KT3 with 50% inhibitory concentration of 6 and 20 nM, respectively. Moreover, the 50% inhibitory concentration of pyronaridine was lower than that of primaquine which is the only drug used to treat malaria patients harboring gametocytes. Prokaryotic (norfloxacin) and eukaryotic (amsacrine and etoposide) DNA topoisomerase II inhibitors were only effective against asexual but not sexual stages of the malaria parasites. Pyronaridine has both schizontocidal and gametocytocidal activities against the human malaria parasite, P. falciparum.

Large scale culture technique for pure Plasmodium falciparum gametocytes.

A large scale technique for pure gametocyte cultures of Plasmodium falciparum was established in culture flasks by using treated erythrocytes and RPMI medium supplemented with 15% human plasma instead of serum. Pure gametocyte cultures were successfully obtained following treatment with 5% sorbitol on day 8 and 9 of cultivation. This method resulted in approximately 97% reduction of asexual parasites and provided pure gametocytes in culture. The highest numbers of gametocytes were obtained from cultures starting with 2% parasitemia and 2% erythrocyte suspension. On day 12 of cultivation, approximately 35 x 10(6) gametocytes per 100 ml of cell suspension could be harvested.

Development of cultivation technique for pure Plasmodium falciparum gametocytes.

Pure gametocyte culture of Plasmodium falciparum, isolate KT3, from Kanchanaburi Province, Thailand, was successfully established by 5% sorbitol treatment on days 9, 10 and 11 following initiation of culture. Using medium supplemented with 15% human plasma and activated erythrocytes, daily medium change was not required during the cultivation. There was 99% reduction in the numbers of asexual parasites in the culture but the numbers of gametocytes were not affected. Furthermore, the gametocytes could undergo their usual morphological development with retention of function as demonstrated by the appearance of exflagellating microgametocytes, macrogametocytes and of oocyst formation in midgut of infected mosquito.