Abstract:

Objective To study the potentiation of chloroquine activity and mechanism by ketotifen and cyproheptadine in in vitro cultured Plasmodium falciparum Fcc SM1/yN strain. Methods In vitro cultured Fcc SM1/yN strain was added to pre-prepared drug plates at 50 μl/well after synchronization to make final concentration of 0.312 5-2 560 nmol/L for chloroqine and of 9.80-5 000 nmol/L for ketotifen or cyproheptadine. After 34 hours’ culture in 37 ℃，the number of schizonts with 3 or more nuclei was calculated among 200 parasites under microscope. Calculated half inhibitive concentration (IC₅₀) of chloroquine and every drug combination to parasite as well as chloroquine activity enhancement index (AEI) of ketotifen (or cyproheptadin). Time dependency of potentiation was studied. All data were analyzed statistically with SPSS 13.0. After 20 hours’ action of one optimal combination dose of chloroquine/ketotifen or chloroquine/cyproheptadine，RNA of the Fcc SM1/yN strain was extracted and real-time PCR was used to determine the expression level of pfcrt and pfmdr1 genes. Results The best potentiation effect was observed with ketotifen or cyproheptadine of 625 nmol/L，with IC₅₀ of 74.53 nmol/L for chloroquine/ketotifen and 89.7 nmol/L for chloroquine/cyproheptadine respectively，and activity enhancement index (AEI) of 0.42 for chloroquine/ketotifen and 0.30 for chloroquine/cyproheptadine respectively. Combination of 625 nmol/L ketotifen or cyproheptadine with 5 nmol/L chloroquine showed the highest potentiation potency. 6-7 hours during which ketotifen or cyproheptadine was added after chloroquine showed the highest effect，with IC₅₀ of 67.70 nmol/L for chloroquine/ketotifen and 81.53 nmol/L for chloroquine/cyproheptadine respectively，and the AEI was 0.47 for chloroquine/ketotifen and 0.37 for chloroquine/cyproheptadine respectively. After action of chloroquine/ketotifen or chloroquine/cyproheptadine at one optimal combination dose，expression level of pfcrt gene increased by 91% and that of pfmdr1 gene decreased by 14% respectively. Conclusion Appropriate combination of chloroquine/ketotiphen or chloroquine/cyproheptadine potentiates chloroquine against in vitro cultured P. falciparum. 6-7 hour period is an optimal time when ketotifen or cyproheptadine was added after chloroquine. Potentiating activity of ketotifen and cyproheptadine may be related to the expression level of pfcrt and pfmdr1 genes.

Key words: Plasmodium alciparum, Resistance, Chloroquine, Ketotifen, Cyproheptadine, pfcrt gene, pfmdr1 gene, Gene expression