Inhibitory activity of ferroquine, versus chloroquine, against western Kenya Plasmodium falciparum field isolates determined by a SYBR Green I in vitro assay

Abstract

Ferroquine (FQ), a chloroquine (CQ) analog, is being developed to treat persons with Plasmodium falciparum malaria. In 146 P. falciparum field isolates from western Kenya, we measured 50% inhibitory concentrations (IC(50); nM) of CQ and FQ by a SYBR Green I in vitro assay. Reference clones included W2 (CQ resistant) and D6 (CQ sensitive). Mutation analysis was done for P. falciparum CQ-resistance transporter gene (Pfcrt K76T). Median IC(50) values for FQ were lower than CQ for field isolates and the W2 clone (both P < 0.05). The Pfcrt mutation (76T), which was detected in > 80% of isolates, conferred higher CQ IC(50) values (P < 0.05) and modestly lower FQ IC(50) values (P < 0.05), versus Pfcrt wild type (K76). FQ is more potent than CQ against CQ-resistant P. falciparum field isolates and the W2 clone, and is less affected by Pfcrt 76T. These findings support the notion that FQ could be useful in treating persons with P. falciparum malaria.

Figure 1.

Chloroquine (CQ) and ferroquine (FQ) SYBR Green I in vitro drug 50% inhibitory concentration (IC₅₀) assays for Plasmodium falciparum field isolates and P. falciparum laboratory reference clones D6 and W2 (right). Individual and median values (closed and open symbols, blue and red bars [online version], and black and gray bars [print version]), respectively are shown. IEV = immediate ex vivo; CA = culture-adapted. Comparison of CQ versus FQ medians within three subsets: P. falciparum field isolates IEV Kisumu, CA Kisii-Kericho, and P. falciparum reference clones D6 versus W2 (all P values < 0.05 by Mann-Whitney rank sum test). Black solid line at 141 nM indicates CQ IC₅₀ historical breakpoint value for H-hypoxanthine assays in P. falciparum field isolates, which is considered discriminative for resistance in vivo.

Figure 2.

Pearson's correlation coefficients of the log 50% inhibitory concentration (IC₅₀) values of chloroquine (CQ)-immediate ex vivo (IEV) versus ferroquine (FQ)-IEV values, and CQ-culture-adapted (CA) versus FQ-CA values. Values were –0.001178 and –0.0596, respectively, which indicated no association (possible cross-resistance) between in vitro activity of CQ and FQ.

Figure 3.

A, Chloroquine (CQ) and B, ferroquine (FQ) 50% inhibitory concentration (IC₅₀) values, respectively, plotted against Plasmodium falciparum chloroqine resistance transporter (Pfcrt) gene wild, mixed, and mutant genotypes. Individual and median 76T values (closed symbols/black bars, and open symbols/gray bars, respectively) are shown. a, P. falciparum field isolates expressing Pfcrt mutation (K76) associated with higher CQ IC₅₀ median values (wild versus mixed versus mutant; P < 0.05, by Kruskal-Wallis one-way analysis of variance (ANOVA) on ranks; wild versus mutant; P < 0.05, by Dunn's method). b, P. falciparum field isolate Pfcrt genotype showed little effect on FQ IC₅₀ median values (gray bars); (wild 76T versus mixed versus mutant; P > 0.05, by Kruskal-Wallis one way ANOVA on ranks). c, P. falciparum field isolates expressing the Pfcrt mutation (K76) associated with higher median IC₅₀ for CQ, versus FQ (closed symbols/black bar and open symbols/gray bar, respectively); (P < 0.05, by Mann-Whitney rank sum test).

Figure 4.

A, Plasmodium falciparum multidrug resistance 1 (Pfmdr1) gene copy number in P. falciparum field isolates and two reference clones. Individual and median values (gray bars) are shown. Several P. falciparum field isolates with slightly higher copy number expression (outliers) were from different sites and had chloroquine (CQ) and ferroquine (FQ) 50% inhibitory concentration (IC₅₀) values within their respective group range. B, CQ and FQ IC₅₀ respectively, plotted against Pfmdr1 codon 86 wild versus mutant genotypes. Individual and median values (closed symbols/black bars, open symbols/gray bars) are shown. For CQ and FQ, Pfmdr1 codon 86 genotype (wild versus mutant) had little effect on IC₅₀ median values (P > 0.05, by Mann-Whitney U test, for CQ [wild versus mutant] and FQ [wild versus mutant]).